
==== Front
101573691
39703
Cell Rep
Cell Rep
Cell reports
2211-1247

38889005
10.1016/j.celrep.2024.114377
nihpa2011865
Article
Antibody-activation of connexin hemichannels in bone osteocytes with ATP release suppresses breast cancer and osteosarcoma malignancy
Riquelme Manuel A. 17
Wang Xuewei 17
Acosta Francisca M. 1
Zhang Jingruo 1
Chavez Jeffery 1
Gu Sumin 1
Zhao Peng 5
Xiong Wei 5
Zhang Ningyan 5
Li Guo 2
Srinivasan Saranya 2
Ma Chaoyu 2
Rao Manjeet K. 34
Sun Lu-Zhe 4
Zhang Nu 26
An Zhiqiang 5*
Jiang Jean X. 18*
1 Departments of Biochemistry and Structural Biology, Microbiology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
2 Immunology & Molecular Genetics, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
3 Greehey Children’s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
4 Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA
5 The Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA
6 South Texas Veterans Health Care System, San Antonio, TX 78229, USA
7 These authors contributed equally
8 Lead contact
AUTHOR CONTRIBUTIONS

Conceptualization, M.A.R., J.X.J., and Z.A.; methodology, M.A.R., X.W., J.Z., J.C., S.G., W.X., N.Z., G.L., S.S., and C.M.; formal analysis, M.A.R., X.W., and N.Z.; investigation, M.A.R., X.W., and P.Z.; resources, Z.A.; writing – original draft, M.A.R., J.X.J., F.M.A., and Z.A.; writing – review & editing, M.A.R., X.W., F.M.A., J.Z., J.C., S.G., W.X., N.Z., G.L., S.S., C.M., M.K.R., L.-Z.S., N.Z., Z.A., and J.X.J.; funding acquisition, J.X.J. and Z.A.; supervision, J.X.J. and Z.A.

* Correspondence: zhiqiang.an@uth.tmc.edu (Z.A.), jiangj@uthscsa.edu (J.X.J.)
26 8 2024
23 7 2024
17 6 2024
07 9 2024
43 7 114377114377
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody’s inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.

In brief

Riquelme et al. report that the M2 antibody opens Cx43 hemichannels in bone osteocytes, releasing ATP. This activates P2X7R, inhibiting cancer cell growth and migration, as well as metastasis of breast cancer and osteosarcoma. Additionally, it enhances anti-tumor immunity by elevating tumor-infiltrating T effector cells and reducing immune-suppressive Treg cells.

Graphical abstract
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pmcINTRODUCTION

Bone metastasis is a leading complication, affecting around 70%–80% of patients with advanced breast cancers, significantly diminishing their quality of life.1,2 Additionally, osteosarcoma, commonly observed in teenagers and young adults and originates in the bone, exhibits poor survival rates in advanced stages due to a lack of effective therapeutics.3 Bone is the most common distal site for breast cancer metastasis, primarily due to its microenvironment characterized by a rich vasculature, abundance of growth factors, cell adhesion molecules, and chemoattractants released by cells within the bone matrix.4,5 Bone tissue comprises three major cells: osteocytes, osteoblasts, and osteoclasts. Osteocytes encompass over 95% of total bone cells and play an essential role in coordinating the activities of osteoblasts and osteoclasts, maintaining bone homeostasis, and remodeling.6–8 Breast cancer bone metastasis is characterized by osteolysis and enhanced osteoclast activity.5

Connexin (Cx) 43, a protein that forms both gap junctions and hemichannels, is highly expressed in osteocytes. Gap junctions facilitate intercellular communication among neighboring cells, while hemichannels enable the passage of molecules between cells and the extracellular environment.9,10 These Cx-forming channels enable the passage of molecules smaller than 1.2 kDa, such as ATP, cyclic AMP, and inositol triphosphate.11,12 Cx43 hemichannels in osteocytes can be activated by alendronate,13 a bisphosphonate drug commonly used for treating bone loss and metastases.14 Furthermore, Cx43 hemichannels mediate the release of small molecules, including ATP and prostaglandins, in response to mechanical loading.15–17

The bone microenvironment provides a supportive environment for breast cancer metastasis. The bone marrow serves as a secondary lymphoid organ with an immunosuppressive microenvironment, abundant in immunosuppressive lymphocytes, such as regulatory T cells (Tregs), which promote and nurture the growth of metastatic tumor growth.18–20 However, the inherent anti-neoplastic properties of bone cells have received limited research attention.21 Moreover, previous studies have not documented the involvement of Cx43 channels in host tissues and their impact on cancer cell proliferation, migration, and metastasis. Recently, we reported that the activation of Cx43 hemichannels in osteocytes, induced by bisphosphonate treatment or mechanical stimulation, suppressed breast cancer cell migration, invasion, and growth.22 The increased release of ATP by active hemichannels is primarily responsible for this inhibitory effect, and the inhibition is mediated through the activation of purinergic receptor signaling in breast cancer cells.23 Additionally, extracellular microenvironments rich in ATP promote the activation of key modulatory cells of the immune system, including antigen-presenting cells, dendritic cells, and macrophages. This process facilitates the recruitment and activation of cytotoxic T (Tc) lymphocytes while reducing the differentiation of T helper cells (Th) into Treg lymphocytes.24–28 These findings suggest that the activation of osteocytic Cx43 hemichannels holds promise as a potential therapeutic strategy for treating breast cancer bone metastasis.22

In this study, we present the generation of a monoclonal antibody, Cx43-M2, that binds to the second extracellular loop of Cx43 and activates Cx43 hemichannels in osteocytes. Additionally, Cx43-M2 can be directly delivered to osteocytes and activates hemichannels in situ. Cx43-M2 induced the opening of Cx43 hemichannel in osteocytes, resulting in ATP release. Furthermore, the activation of P2X7 receptor, triggered by this ATP release, suppressed breast cancer cell growth and migration. Cx43-M2 effectively inhibited breast cancer growth in mouse breast carcinoma in wild-type (WT) mice and human breast cancer cells in immune-compromised mice in vivo. When administered to immunocompetent mice, Cx43-M2 increased the relative abundance of tumor-infiltrating Tc and Th lymphocytes, while concurrently decreasing the levels of immune-suppressive Treg cells. Taken together, this study highlights the potential for a therapeutic approach based on a unique mechanism of action.

RESULTS

Development of an antibody that activates Cx43 hemichannels

We generated several mouse hybridoma monoclonal antibodies against the second extracellular domain of Cx43 (Figure 1A). After testing their binding and hemichannel activities, we identified a potent clone, Cx43-M2, which effectively activates Cx43 hemichannels. The antibody-encoding genes from the hybridoma cell line were cloned and engineered into a mouse/human chimeric immunoglobulin G1 (IgG1) named Cx43-M2(MH) and a humanized IgG1 named Cx43-M2(H). Cx43-M2(MH) and Cx43-M2(H) were expressed transiently in HEK293 cells. The immunoreactivity of Cx43-M2(MH) and Cx43-M2(H) against Cx43 was further confirmed in HeLa-Cx43 cells, which stably express Cx43, but not in parental HeLa cells (Figure 1B). We detected Cx43 on the cell surface of osteocytic MLO-Y4 cells (Figure 1C). The cell surface was counterstained with wheat germ agglutinin (WGA) (red), which binds to glycoproteins present in the cell surface (Figure 1C). We determined the binding affinities of the Cx43-M2(H) antibody to the Cx43 M2 peptide by bio-layer interferometry. As shown in Figure 1D, the Kd for the Cx43 M2 peptide was 89 nM for Cx43-M2(H).

Cx43-M2(MH) dose dependently increased Cx43 hemichannel-mediated dye uptake in MLO-Y4 osteocytes (Figure 2A). Because the antibody binds to the extracellular side of Cx43, there was a chance that this antibody could interfere in the gap junction communication between cells. To assess this, intercellular coupling was tested using a scrape wound dye transfer assay (Figure 2B, right) for various periods (Figure S1). MLO-Y4 cells were coupled, and as expected, the exposure of the cells to carbenoxolone (CBX), a Cx channel inhibitor, significantly inhibited the dye transfer (Figures 2B and S1). However, the presence of the antibody did not block gap junction cell coupling after incubating with M2(MH) for up to 24 h (Figures 2B and S1). We further validated this observation using a noninvasive, parachuting dye transfer assay to assess gap junctions29 using MLO-Y4 osteocytes and BT474 breast cancer cells expressing Cx43. The results showed that MLO-Y4 cells formed gap junction channels between themselves (Figure S2A), consistent with previous studies,30 but not with cancer cells, regardless of whether MLO-Y4 cells or BT474 cells served as donor or recipient cells (Figures S2B and S2C). We could not detect any dye transfer between BT474 breast cancer cells (Figure S2D). This result suggests that the antibody did not affect the docking of the Cx43 hemichannels required to form functional gap junction channels. The activation of hemichannels by both Cx43-M2(MH) and Cx43-M2(H) was also observed in primary osteocytes isolated from mouse long bones, but not by human IgG control (Figures 2C and 2D). Additionally, 10Panx, a specific pannexin 1 hemichannel blocker, did not affect the dye uptake induced by M2(MH) (Figure S2E), which indicates the specific activating effect of Cx43-M2(MH) on Cx43 hemichannels. Moreover, the opening of Cx43 hemichannels is known to release ATP.22,31 Cx43-M2(MH) increased the extracellular ATP released from MLO-Y4 osteocytes. This release was inhibited with treatment CBX (Figure 2E). Transwell migration assay showed that the conditioned media (CM) collected from MLO-Y4 cells treated with Cx43-M2(MH) resulted in a reduction in breast cancer cell migration (Figure 2F). CM collected from Cx43-M2(MH) also inhibited breast cancer cell growth by suppressing spheroid formation (Figure S3A) and increased cancer cell death (Figure S3B). However, Cx43-M2(MH) did not affect cell migration when we directly treated MDA-MB-231 cells with the antibody (Figure 2G). These data suggest that Cx43-M2 has no direct effect on cancer cells, and the suppression of cancer cells is mediated by activating Cx43 hemichannels and the factors released from osteocytes.

Given that the antibody activates Cx43 hemichannels, and active osteocytic hemichannels inhibit breast cancer bone metastasis,23 we aimed to assess the efficacy of the antibody in vivo. We intraperitoneally (i.p.) injected 25 mg/kg Cx43-M2(MH), and the injected antibody was detected on the surface of osteocytes in cortical bones (Figure 3A). This result demonstrates the delivery of the antibody to Cx43 hemichannels in bone osteocytes in vivo. Next, we evaluated the activity of Cx43-M2(MH) in osteocytes in situ, using an in vivo dye uptake assay. Mice were i.p. injected with 25 mg/kg Cx43-M2(MH) 6 h before the intravenous injection with 200 mg/kg Evans blue dye. After 30 min of incubation, perfusion, and fixation, bone sections were stained with DAPI for nuclear staining. The cortical and trabecular bone from mice exposed to Cx43-M2(MH) showed significantly more Evans blue dye uptake (red) compared to the saline control (Figure 3B), confirming the stimulatory effect of the antibody on osteocytic Cx43 hemichannel opening in both cortical and trabecular bones in vivo. Together, we demonstrate that the antibody that binds specifically to Cx43 induces the opening of hemichannels with an increase in ATP release from osteocytes, the most abundant cell type in the bone.

The Cx43-M2 antibody reduces breast cancer growth and metastasis in the bone

We next investigated the impact of the Cx43 hemichannel-activating antibody, Cx43-M2(MH), on breast cancer bone metastasis. Triple-negative MDA-MB-231 human breast cancer cells were implanted into mouse tibias of athymic mice, and the anti-body was administrated weekly via i.p. injection. Treatment with the antibody at a dosage of 25 mg/kg resulted in the inhibition of tumor growth, as indicated by reduced bioluminescence signals and tumor size (Figure 4A). The antibody-treated group exhibited significantly reduced tumor growth compared to the vehicle (saline) and human IgG (hIgG) control groups after 6 and 7 weeks of treatment (Figure 4B).

To further assess the potency of Cx43-M2(MH) in vivo, we conducted intratibial implantation of murine PY8119 triple-negative breast cancer cells in WT-C57BL/6 mice, with or without antibody treatment. Notably, Cx43-M2(MH) significantly suppressed PY8119 breast cancer growth in the long bone (Figure 4C). We also examined the dosing frequency of the antibody and observed a consistent suppressive effect when mice were treated either once or twice per week with a dosage of 25 mg/kg Cx43-M2(MH) after 4 weeks (Figure 4D). Furthermore, we investigated the dosage response by treating mice once per week with varying dosages of Cx43-M2(MH) (5, 15, and 25 mg/kg) for 4 weeks using the PY8119 tumor tibial implantation model (Figure 4E). The results demonstrated a significant decrease in tumor size at dosages of 15 and 25 mg/kg at 4 weeks, while the 5-mg/kg dosage had minimal effects. Moreover, treatment with Cx43-M2(MH) at 25 mg/kg for 4 weeks effectively protected bone against breast cancer-induced osteolysis and bone damage, as observed through X-ray imaging (dashed circles, Figure 4F). To investigate the specificity of Cx43-M2(MH) for osteocytic Cx43 hemichannels, we utilized osteocyte-specific conditional knockout (cKO) mice (OstCx43KO) driven by a 10-kb promoter.22 Compared with the WT control, we found that the inhibitory effect of Cx43-M2(MH) on breast cancer bone metastasis was abrogated in cKO mice (Figure 4G). These data further support the notion that the tumor suppressive effect of M2(MH) requires osteocytic Cx43 as the molecular target.

To avoid immunogenicity in clinical development, we humanized the murine antibody by a complementarity determining regions grafting protocol, as described previously.32 The efficacy of humanized Cx43-M2(H) was assessed across different subtypes of human breast cancer cells in athymic mice, following a dose response. Similar to the mouse-human chimeric Cx43-M2(MH) antibody, the humanized Cx43-M2(H) antibody also targeted osteocytes in vivo, and positive signals were absent in the liver, heart, and spinal cord, the tissues known to be enriched inconnexins (Figures S3C and S3D). Weekly i.p. administration of Cx43-M2(H) at 25 mg/kg significantly inhibited tumor growth of triple-negative human MDA-MB-231 breast cancer cells using an intratibial implantation tumor model. However, dosages of 15 and 5 mg/kg showed some reduction in tumor growth, but not at a statistically significant level (Figure 4H). In athymic female mice intratibially implanted with Her2-BT474, a human luminal A, ER+/PR+/HER2+ breast cancer cell line, weekly treatment of Cx43-M2(H) at 25 mg/kg inhibited the intratibial growth, while lower dosages were ineffective (Figure 4I). Additionally, treatment of athymic mice with intratibially implanted T47D, a human luminal A ER+/PR+/HER2− breast cancer cell line, exhibited a dose-dependent inhibition of tumor growth, and the efficacy of Cx43-M2(H) in suppressing tumor growth was significant at all tested concentrations after 6 weeks (Figure 4J). The significant inhibition was consistently observed at 25 mg/kg after 4 weeks of treatment across various subtypes of breast cancer (Figures S4A–S4C). These findings provide evidence of the effectiveness in inhibiting tumor growth retained in the humanized antibody in multiple breast cancer subtypes in a dose-dependent manner.

The question is why activation of osteocytic hemichannels by Cx43-M2 suppresses tumor growth. Cx43 hemichannels mediate the release of ATP directly,33 and ATP is tumor suppressive.23,34 To investigate whether the inhibitory effect of Cx43-M2(MH) on breast cancer cell migration was mediated through ATP release, CM was collected from MLO-Y4 osteocytes treated with or without 25 mg/kg M2(MH), an ecto-ATPase inhibitor (ARL61756) or an ATP-degradation enzyme (apyrase [Apy]). The CM was then used to treat PY8119 cells and assess cell migration (Figures 5A and 5B). Treatment with CM from Cx43-M2(MH)-treated MLO-Y4 cells significantly inhibited cell migration; this inhibition was further enhanced with pre-treatment with ARL61756 (Figure 5A). Conversely, the inhibitory effect of Cx43-M2(MH) was attenuated with Apy (Figure 5B). ATP in naive medium at lower concentrations (50 μM) inhibited the migration of MDA-MB-231 cells, while higher concentrations (100 and 200 μM) of ATP exhibited a trend toward increased migrated cell numbers (Figure S5A), due to the degradation of ATP into its by-product, adenosine, which promotes tumor growth.23 We have previously shown that ATP exerts a tumor inhibitory function by activating the P2X7 receptor (P2X7R).23 The inhibitory effect of CM collected from Cx43-M2(MH)-treated osteocytes on the migration of MDA-MB-231 cells was attenuated when the cancer cells were treated with a P2X7R antagonist, oxidized ATP (oATP) (Figure S5B). The activation of P2X7R mediates Ca2+ influx and elevates intracellular Ca2+ signaling.35,36 P2X7R agonist 3′-O-(4-benzoyl)benzoyl ATP (BzATP) increased intracellular Ca2+ levels in MDA-MB-231 cells, and this increase was attenuated by the P2X7R antagonists oATP and A804598 (Figure S5C). Reduction of MDA-MB-231 cell migration by BzATP, as shown in a transwell assay, was significantly attenuated by the P2X7R antagonist A804598 and the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP) II (Figure S5D). Moreover, the increased microtubule (MT) polymerization induced by BzATP was attenuated by the P2X7R antagonist A804598 and the CaMKII inhibitor AIP (Figure S5E). These results indicate that increased Ca2+ signaling by the P2X7R is inversely correlated with cell mobility, and activation of CaMKII is likely to be involved. Cell mobility is driven by the cooperation of MTs and stathmin, an oncogene that depolymerizes MTs through interaction with α/β-tubulin, thus promoting cell mobility.37 Additionally, we compared P2X7R mRNA levels between normal breast tissues and breast cancer tissues in The Cancer Genome Atlas (http://cancergenome.nih.gov/) and found a significant reduction in P2X7R mRNA levels in breast cancer tissues (Figure S5F). The results suggest that ATP released by Cx43-M2-activated hemichannels in osteocytes inhibits breast cancer cell migration through the action of the P2X7R signaling pathway in cancer cells.

Bone is one of the largest endocrine organs; osteocytes comprise over 90%–95% of total bone cells. It is plausible that ATP released by osteocytic Cx43 hemichannels may have a major impact on ATP level in the circulation. We assessed ATP levels in the plasma collected from the mice treated with Cx43-M2(MH) for various periods (6, 24, and 48 h) or at varying dosages (5, 15, and 25 mg/kg). The administration of 25 mg/kg Cx43-M2(MH) increased the plasmatic ATP levels in a time-dependent manner, with a maximal accumulation at 24 h (Figure 5C). Cx43-M2(MH) also significantly elevated plasmatic ATP levels in a dose-dependent manner, with a greater level of ATP with 25 mg/kg (Figure 5D).

Given the elevated levels of tumor-inhibitory ATP in the circulation, we further investigated the impact of the antibody on breast cancer growth and metastasis in distal regions outside the bone using an intracardiac injection cancer metastasis model.38,39 We intracardiacally injected human MDA-MB-231 and murine PY8119 cells into athymic (Figure 5E) and WT C57BL/6 mice (Figure 5G), respectively. Weekly treatment with 25 mg/kg Cx43-M2(MH) reduced tumor growth and metastasis, as indicated by the photon flux signals and areas, respectively (Figures 5E and 5G). Cx43-M2(MH) significantly increased the survival rates of athymic and WT mice bearing breast cancer (Figures 5F and 5H). To account for the aforementioned differences in cell lines (murine vs. human breast cancer cells) and environments (C57BL/6 vs. athymic), we injected the same murine PY8119 cells by intracardiac injection into athymic mice and found that compared to its effect in C57BL/6 mice, there was no significant difference in survival. However, Cx43-M2(MH) retained its ability to reduce cancer metastatic spread significantly (Figure S6). These findings indicate that Cx43-M2(MH) exerts its tumor inhibitory effect through its role in osteocytic ATP release, and the elevated ATP in the body likely suppresses tumor growth and metastasis not only locally (bone) but also systemically. Additionally, T cells activated by Cx43-M2 antibody are likely needed for enhancing survival rates but have less effect on reducing tumor dissemination.

The Cx43-M2 antibody promotes adaptive anti-tumor immunity in lymph nodes and infiltrating tumor tissues

ATP is known to act as an immune activator, promoting immune responses.24–28,40,41 We tested the hypothesis that the effect of the Cx43-M2 antibody on breast cancer might be partly due to ATP-mediated enhanced anti-tumor immunity. To evaluate changes in the lymphocyte population in mice treated with the Cx43-M2(MH) antibody, we performed flow cytometry analysis on lymph node, plasma, and tumor samples using antibodies specific to protein markers for various T lymphocytes.42,43 The flow gates were set to detect the relative amount of T lymphocytes to GFP− non-tumor cells, as tumor cells expressed GFP. Considering the pivotal role of T lymphocytes in immune adaptation and their function as indicators of the tumor environment, we assessed the level of lymphocytes in the draining inguinal lymph nodes.44 Representative flow cytometry data are shown in Figure 6A. Quantification analysis revealed a significant increase in the number of immune-promoting effector T cells, CD8+ Tc (CD3+/CD8+), and CD4+ helper T lymphocytes (Th, CD3+/CD4+) following weekly treatment with Cx43-M2(MH) for 4 weeks compared to the saline group in WT C57BL/6 mice with tibial implantation of PY8119 cells (Figure 6B). However, Cx43-M2(MH) did not affect the levels of immune-suppressive Tregs (CD3+/CD4+/CD25+). Notably, M2(MH) did not induce significant changes in the three types of T lymphocytes in mice without tumors. M2(MH) treatment did not affect the levels of circulating Tc and Tregs, but it did increase circulating Th lymphocytes in tumor-bearing mice (Figure 6C). Finally, tumor-infiltrating lymphocytes, especially Tc and Th lymphocytes, were augmented after treatment with Cx43-M2(MH) compared to saline. Conversely, Tregs in mice treated with Cx43-M2(MH) were significantly lower than those in saline-treated mice (Figure 6D). The reduction of Tregs in infiltrating tumor tissue was further confirmed with a significant decrease in Foxp3+ cells after Cx43-M2(MH) treatment (Figure S7A). Moreover, the antibody treatment significantly decreased immune-suppressive myeloid-derived suppressor cells in mouse bone marrow (Figure S7B). Additionally, the cytotoxic CD8+ T cells in tumor-infiltrating lymphocytes were activated as indicated by a significant increase in granzyme A (Gzma) and programmed cell death protein 1+ (PD-1+)cells (Figure 6D). Collectively, Cx43-M2(MH) treatment promotes anti-tumor immunity by enhancing immune-promoting T lymphocytes in draining lymph nodes and tumor tissue, while inhibiting tumor-suppressive infiltrating Tregs in tumor tissue. The ability to reduce Tregs in tumor tissues has the potential to compensate for the ineffectiveness or adverse effects of current immunotherapies.45

The efficacy of Cx43-M2(MH) to inhibit tumor growth was reduced by P2X7R antagonist Brilliant blue G (BBG) (Figure S7C). The increase in Tc and Th in draining lymphocytes by M2(MH) showed a trend of reduction with the BBG (Figures S7D, and S7E) and did not affect Treg lymphocytes (Figure S7F). This further shows that ATP tumor inhibitory function through the activation of the P2X7R can be driven through lymphocyte modulation.

Cx43-M2 antibody via ATP release effectively inhibits osteosarcoma growth and metastasis

Given that ATP released by osteocytic Cx43 hemichannels generates a tumor-suppressive bone microenvironment against breast cancer, we investigated the effectiveness of the antibody in osteosarcoma, cancer originating in the bones. Similar to its inhibitory effect on breast cancer cells, CM collected from Cx43-M2(H)-treated osteocytes significantly reduced the migration of human OS17 osteosarcoma cells, as determined by a cell transwell migration assay. However, this inhibitory effect was completely attenuated when the CM was incubated with Apy, an enzyme that hydrolyzes ATP (Figure 7A). We further confirmed the role of ATP on OS17 cells by observing a significant inhibition of tumor growth when treated with ATPgS, an ATP analog (Figure 7B). These findings strongly suggest that ATP released from osteocytes is responsible for inhibiting tumor growth in human osteosarcoma cells.

The efficacy of the Cx43-M2 antibody was assessed in vivo by intratibial injection of multiple human and murine osteosarcoma cell lines (Figures 7C–7F). The inhibitory effects were observed in human osteosarcoma OS17 and 143B cells, where tumor growth suppression was significant and dose dependent (Figures 7C and 7D). Weekly treatment with M2(MH) also significantly reduced DLM-8 murine osteosarcoma growth and metastatic coverage in syngeneic mice (Figures 7Eand 7F). Cx43-M2(MH) also significantly increased the survival of mice implanted with murine DLM8 osteosarcoma cells at 15 and 25 mg/kg, depicted using a Kaplan-Meier curve (Figure 7G). These observations suggest that the activation of Cx43 hemichannels by the antibody transforms the bone from tumor supporting to tumor suppressive, effectively reducing osteosarcoma growth and metastasis and increasing survival rates.

DISCUSSION

We have previously reported that the activation of Cx43 hemichannels inhibits breast cancer bone metastasis in vivo.22 This inhibition is achieved by releasing ATP, which activates growth-inhibitory purinergic signaling in breast cancer cells.23 Given that Cx43 hemichannels in osteocytes are typically closed,46 pharmacological activation of these channels would be a promising strategy for treating metastasized cancer in the bone. Most of the reagents, including chemical compounds, peptides, and antibodies, that target connexin channels are inhibitory reagents designed to inhibit hemichannels or/and gap junctions.47 The Cx43-M2 antibody targets the second extracellular domain of Cx43. This domain has been used previously as a target to manipulate the activity of Cx43 hemichannels, primarily with the inhibitory reagents, as seen with the polyclonal Cx43 antibody (Cx43E2) and humanized mouse antibody close 1 (MHC1) that we have developed for treating spinal cord injury,16,46,48 as well as peptides, Gap27, and P180–195.47 Our study marks the development of a connexin-activating antibody. Unlike Cx channel targeted peptides, this antibody exhibits high specificity for Cx43 hemichannel rather than gap junctions. Even after 16 h of the treatment, gap junction intercellular communication remains unaffected. The Cx43-M2 antibody binds to the second extracellular domain of Cx43, and this binding does not appear to affect gap junctions, as evidenced by another Cx43 hemichannel-blocking antibody, MHC1, that we developed previously.48 This observation suggests that the epitope and domain of interactions with the antibody may not participate in docking hemichannels to form gap junction channels. Studies have indicated that hemichannels are sorted into specialized domains in the plasma membrane when they are to be docked with another hemichannel, which may not be reachable to the antibody.48,49 Alternatively, it is possible that bulky antibodies are not capable of penetrating tightly packed gap junctional plaques, especially in the presence of large clusters of detergent-resistant gap junction particles.50

By analyzing various organs with a rich presence of Cx43, we found that the Cx43-M2 antibody is primarily delivered to the surface of bone osteocytes in situ. Distinctively, no detectable signals were observed in the heart, which expresses Cx40, Cx43, Cx45, and Cx37; the liver, which expresses Cx43, Cx26, and Cx32; and the spinal cord, which expresses Cx43, Cx32, and Cx30.51 This indicates that the Cx43-M2 antibody specifically targets bone osteocytes. The absence of Cx43-M2 anti-body in the spinal cord is expected because antibodies cannot typically cross the blood-brain or spinal cord barrier. The lack of the signal in Cx43-rich organs could be explained by the fact that Cx43 primarily forms gap junctions, not hemichannels, in these tissues,52 and the Cx43-M2 antibody does not affect gap junctions.

Bone is the preferred site for breast cancer metastasis, particularly for ER+/Her2− breast cancer. Notably, our results indicated that ER+/Her2− T47D cells were the most responsive to M2 antibody treatment compared to other breast cancer subtypes, where significant inhibition could be observed even at a lower dosage of 5 mg/kg. The specificity of the antibody targeting Cx43 in osteocytes was further demonstrated using an osteocyte-specific Cx43 cKO mouse model. The lack of effect of the Cx43-M2 antibody in osteocyte-specific cKO confirms that tumor inhibition is due to the activation of Cx43 hemichannels in osteocytes, not tumor cells. Interestingly, we did not observe an increase in tumor growth in the Cx43 cKO compared to the previous study.22 This difference could be related to the number of cancer cells we injected. The number of cells injected to initiate this process may impact the tumor microenvironment. In our previous paper, we injected 100,000 cells,22 while in the present study, we injected 20,000 cells because the growth pattern was consistent with this cell number for other in vivo studies conducted here. The implantation of a larger number of cancer cells, 100,000 vs. 20,000, revealed greater tumor growth in a microenvironment with poor bone quality due to osteocyte Cx43 deletion. Compared to WT mice, there is a major shift in the bone microenvironment in Cx43 cKO mice due to decreased bone quality, leading to varying responses to the number of tumor cells.

Active hemichannel-releasing tumor suppressive factors likely transform the tumor-supportive microenvironment into a tumor-hostile one. Osteocytes constitute over 95% of total bone cells, and hemichannels are abundantly present on their surface, suggesting that the opening of these channels would significantly impact the bone microenvironment. We showed that at the late stage of tumor growth, the tumor lesions appeared to extend beyond the bone cavity. This is because the implanted breast cancer cells grow rapidly and extensively, destroying the bone as well as other tissues from the inside to the outside. After 4 weeks of tumor implantation, where exponential tumor growth is evident, cancer cell numbers surpass osteocytes in proportion. This could compromise the tumor suppressive function of osteocytes. Due to the aggressive growth pattern of this model, we treated mice after cancer cell implantation in the tibia, not at later stages when cancer cells could significantly outnumber osteocytes.

We found that the Cx43-M2-induced ATP in plasma is not time dependent, with higher levels observed after 24 h of treatment than those after 6 and 48 h. ATP is unstable in the body due to cell surface-expressed and released ectoATPases.53 Additionally, the antibody may require more than 6 h to maximize its effects, reaching a maximum at around 24 h in mouse plasma. Considering the very short half-life of ATP in plasma, with just 1% of an ATP bolus remaining after 40 s,54 the release of ATP induced by the antibody may be intense or consistent over time, but not time dependent due to the robust ATP degradation system. It is also possible that the hemichannels and concomitant antibodies are inactivated due to factors such as antibody internalization by osteocytes. However, this seems unlikely because the release of ATP remains concentration dependent in vitro. This biphasic pattern of ATP over time in the plasma could be the end consequences of ATP release and ectoATPase activity. ATP can directly bind to P2X7R, leading to increased intracellular Ca2+ signaling, which activates CaMKII and enhances MT polymerization, a hallmark of inhibited cancer cell migration.55 Our data indicate that the CaMKII/MT axis is a likely mechanism connecting P2X7R-activated Ca2+ signaling with cancer cell mobility.

Additionally, we have previously shown that ATP can activate P2Y11R, which inhibits the expression of CXCR4,34 reducing its binding to CXCL12. This is crucial as CXCR4-CXCL12 binding is known to promote breast cancer migration.56 However, it should be noted that the activation of P2X7R and other purinergic receptors has also been implicated in promoting tumor cell survival and proliferation.57,58 ATP acts as an inhibitory factor on breast cancer cells and functions as an immune activator. Given the elevated ATP levels in the blood of Cx43-M2-treated mice, we anticipated observing the tumor-suppressive function at a global level, beyond just the bone tissues. Indeed, using the intracardiac tumor implantation metastasis model, we demonstrated a reduction in breast cancer metastasis and an increase in survival rate following Cx43-M2 treatment. Cx43-M2 treatment led to increased levels of Tc and Th lymphocytes, the major adaptive immune response against tumors, both in draining lymph nodes and tumor tissue. T lymphocytes play a critical role in anti-tumor immunity.59 In immunocompetent WT mice, administration of the antibody enhanced anti-tumor immunity by increasing infiltrating effector T lymphocytes, specifically CD8+ cytotoxic T cells (Tc), and CD4+ Th cells, while reducing the immune-suppressive subtype of CD4+/CD25+ Tregs. Treg cells generally suppress the induction and proliferation of effector CD8+ cells.60 We further measured the activation of cytotoxic T cells (CD8) in the infiltrating tumor tissue using two markers: Gzma and PD-1. PD-1 is induced by T cell receptor (TCR) signaling and can be used as a surrogate marker to label tumor-specific T cells. Cytotoxic lymphocytes express Gzma. Our data showed that the antibody treatment increased the activation of cytotoxic T cells, as indicated by a significant increase in CD8+ cells positive for both Gzma and PD-1. This finding suggests that the antibody treatment significantly enhances the activation of tumor-targeting cytotoxic T cells.

The effect of Cx43-M2 on the immune landscape is ATP dependent. It is established that extracellular ATP promotes the activation of key immune system modulators, such as antigen-presenting cells, dendritic cells, and macrophages, thus facilitating the recruitment and activation of Tc lymphocytes while reducing the differentiation of Th into Tregs. Additionally, ATP promotes tissue-resident memory T cells (CD8+ Trm) through P2X7R, and Trm has been shown to correlate directly with tumor growth control in both mouse models and human patients.24–28,61,62 Together, the mechanism of tumor suppression by Cx43-M2 appears to act on both the tumor and the immune system through the ATP release by osteocytic Cx43 hemichannels and activation of purinergic receptor signaling. We demonstrated that the antibody also exhibited a tumor-suppressive effect in athymic mice lacking T lymphocytes. However, at this stage, we cannot rule out the participation of other immune cells in the process.

Cx43-M2 also reduces Treg lymphocytes in tumor tissues. Treg cells are immune-suppressive T cells, and their presence and activation in tumor tissues present a major challenge for current immune therapy, including PD-1/PD-ligand 1 therapies.42,63 Cx43-M2 could compensate for the deficits of other immune therapies by suppressing infiltrating Treg cells, and its non-overlapping mechanism of action may improve the efficacy of current immunotherapy by increasing immune cells’ exposure to immune activators such as extracellular ATP.

Limitations of the study

We acknowledge certain limitations in our study, with two major ones being identified. First, in our model, the lack of knowledge about tumor antigens poses a challenge in measuring T cell function using a co-culturing approach, primarily due to the extremely low frequency of tumor-specific T cells. Given the random rearrangement of the TCR gene during T cell development, it is estimated that among all CD4 and CD8 T cells, only about 100–1000 naive T cells recognize a given antigen in a mouse. Considering that an adult mouse typically hosts at least 2 × 107 CD4 and 107 CD8 T cells, the frequency falls well below 1/1,000. Detecting T cell proliferation is unlikely without prolonged in vitro culture/stimulation, which would significantly alter the T cell phenotype. To address this issue, we measured the activation of cytotoxic T cells (CD8) in the infiltrating tumor tissue. However, further effort is needed to understand the purinergic regulation on immune and tumor environment. Second, we used the intratibial injection model to implant tumor cells locally into the bone marrow. This method was chosen because the incidence of bone metastasis via systemic metastasis, such as through intracardiac injection, is very low, making it challenging to evaluate the extent of tumor growth. We used X-rays to determine tumor lesions in the bone and bone osteolysis and luciferase signals to determine the tumor size. Disrupted structures of soft tissue are difficult to detect using X-rays.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jean X. Jiang (jiangj@uthscsa.edu).

Materials availability

All unique/stable antibodies, cells and mice generated in this study will be available and shared by the lead contact upon request, but we may require a completed materials transfer agreement if there is potential for commercial application.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request.

This paper does not report the original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal models

Mice were group housed (5 maximum) in non-environmentally enriched cages with unrestricted food and water access and a 12 h light-dark cycle. Room temperature was maintained at 21 ± 2°C. Animal husbandry was carried out by UT Health San Antonio technical staff. The animal use protocols used in this manuscript were approved by the UT Health San Antonio Institutional Animal Care and Use Committee. Six to seven-week old female C57BL/6 mice were used for the intratibial injections as previously described.23 Mice with floxed Cx43 gene (Cx43flx/flx) were originally generated by Dr. Klaus Willecke’s lab at the University of Bonn, Germany64 and provided by Dr. Roberto Civitelli at Washington University. Mice with a deletion of Cx43 from osteocytes were generated using the Cre/Lox system. Initially, mice with the floxed Cx43 gene (Cx43flx/flx) were crossed with Cx43 heterozygous mice expressing one Cx43 allele (Cx43+/−). Subsequently, mice expressing a Cre recombinase driven by a 10-kb DMP1 promoter, which leads to gene expression predominantly in osteocytes65 (DMP1-Cre; Cx43+/+), were crossed with Cx43fl/− mice to generate Cx43 osteocyte-specific cKO (DMP1-Cre; Cx43fl/−) mice. Genotyping was performed using polymerase chain reaction (PCR) techniques with genomic DNA isolated from mouse tails and corresponding primers synthesized at the UTHSCSA DNA Core Facility.

Cell culture

Murine osteocytic MLO-Y4 cells were seeded on plates coated with rat tail collagen type I (BD Biosciences, San Jose, CA) and grown in α-modified essential medium (α-MEM) supplemented with 2.5% fetal bovine serum (FBS) and 2.5% bovine calf serum (BCS). The murine mammary carcinoma cell lines PY8119 and PY230 were grown in F12K nutrient media (Gibco) supplemented with 5% Fetal Clone II (Fisher Scientific). Human MDA-MB-231, T47D, and BT474 cells were cultured in McCoy’s 5A modified media with 10% FBS. Human osteosarcoma cells 143B and OS17 cells were cultured in D-MEM media with 10% FBS. The murine osteosarcoma cell line DML8 cells were cultured in D-MEM media with 10% FBS. HeLa cells were cultured in D-MEM media with 10% FBS. All cells were grown at 37°C, 5% CO2.

METHOD DETAILS

Generation of monoclonal antibody

A peptide located in the second extracellular loop domain of Cx43 was used by Abmart Co. (Shanghai, China) to generate mouse monoclonal hybridoma. We identified that clone (C) 2 had the potency to activate Cx43 hemichannels. Through PCR and subcloning, we generated a mouse-human chimeric antibody, Cx43-M2(MH), with a human heavy chain and a mouse light chain. Humanization of the antibody was based on a complementarity determining regions (CDR)-grafting strategy as described previously.32 Briefly, CDRs in the heavy and light chains of the mouse antibody were defined by a combination of three methods: Kabat, IMGT, and Paratome. The parental mouse monoclonal antibody (mAb) and the most closely related human germline sequence were then aligned. Residues that are known not to be structurally critical and/or subjected to change during the in vivo maturation process were identified. The human IgG signal peptides and a Kozak sequence were engineered at the 5′-ends of the VL and VH sequences. The humanized VL and VH fragments were then cloned into human IgG1 CL and CH vectors separately. Expression, purification, and quantification of the mAbs are the same as described previously.32 The antibody affinity to the M2 peptide was measured using Bio-Layer Interferometry (BLI) with the Octet RED96 instrument (FortéBio, Fremont, CA). Briefly, the antibodies were loaded onto protein A biosensors at a concentration of 20 μg/mL in 1x kinetic buffer for 5 min. The coated biosensors were then incubated with a series of M2 peptide concentrations (ranging from 0 to 500 nM) for 3 min, followed by a washing step in 1x kinetic buffer for an additional 10 min to allow for dissociation. FortéBio’s data analysis software was employed to fit the binding curve to a 1:1 binding model in order to extract association and dissociation rates. The affinity (KD) was subsequently calculated using the ratio of koff to kon.

Conditioned media (CM) preparation

After 24 h of culture, the media for MLO-Y4 cells was changed to α-MEM without phenol red, supplemented with 2.5% FBS and 2.5% BCS. MLO-Y4 cells were then incubated in the absence or presence of 6 μg/mL Cx43-M2(MH) or Cx43-M2(H) antibody for 48 h, and the conditioned media (CM) was collected. Primary mouse osteocytes were obtained from 4-month-old mice, following sequential decalcifications with EDTA and digestion with collagenase type 1, as described.66

Immunofluorescence

HeLa cells stably transfected with Cx43 were generously provided by Dr. Bruce Nicholson at the University of Texas Health Science Center at San Antonio. Parental and Cx43-transfected HeLa cells were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature (RT), permeabilized with 0.25% Triton X-100, and blocked with 3% BSA. The cells were then incubated overnight at 4°C with Cx43-M2(MH) or Cx43-M2(H) antibody (50 μg/mL) for 1 h, followed by incubation with peroxidase-conjugated anti-mouse or anti-human secondary antibody and staining with the ABC kit (Vector laboratories, Newark, CA, USA). Surface labeling was performed according to our previously published protocol.67 Briefly, MLO-Y4 cells were washed in PBS and then incubated for 1 h with Cx43-M2(MH) or Cx43-M2(H) antibody (5 μg/mL). The cells were fixed in 4% PFA and subsequently labeled with secondary antibodies for 1 h each at RT. Slides were mounted using Heatshield mounting medium (H-1000, Vector Lab). Confocal fluorescence imaging was conducted using a confocal laser scanning microscope (Flu view; Olympus Optical, Tokyo, Japan).

To detect the presence of Cx43-M2(MH) or Cx43-M2(H) antibody in situ, Cx43-M2(MH) or Cx43-M2(H) (25 mg/kg) was injected via the tail vein. Mice were sacrificed 2 h after injection. The mice were perfused with PBS 3 times their volume and then fixed with 4% PFA in PBS. Tibias, liver, and heart were isolated. Tibial tissue sections were decalcified by daily changes of 10% EDTA for 2 weeks. The tissues were embedded in OCT and cut using a cryostat. The presence of antibodies was detected using FITC-conjugated secondary IgG specific to the human IgG heavy chain.

Dye uptake in vitro and in vivo

MLO-Y4 cells were treated with or without 6 mg/mL Cx43-M2(MH) or Cx43-M2(H) for 30 min. Afterward, the cells were incubated with recording medium (α-MEM+10 mM HEPES) containing ethidium bromide (EtBr) 50 μM for 5 min. The cells were then rinsed four times with PBS and fixed with 2% PFA. At least three pictures of fluorescence fields were taken using an inverted microscope (Keyence BX-700, Tokyo, Japan) with a rhodamine filter. The images were subsequently analyzed using NIH ImageJ software. Defined circular regions of interest (ROIs) over the nucleus were used to measure the average pixel density of 30 random cells.

Evans blue dye was injected into the tail vein of 4-month-old mice. Cx43-M2(MH) (25 mg/kg) or saline control was intraperitoneally injected 2 h before the dye injection. After 2 h, the mice were scarified and perfused with PBS to remove excess dye. Tibias were isolated and fixed, and tibial tissue sections were prepared. Dye uptake was measured in the cortical and trabecular bone areas using Evans blue fluorescence and counterstained with DAPI for the nucleus. The extent of dye uptake was quantified by NIH ImageJ software.

Scrape-loading dye transfer assay

Gap junction intercellular coupling was assessed at room temperature (25°C) using the scrape-loading/dye transfer technique in MLO-Y4 cells, in the absence or presence of 30 μg/mL of M2(MH) or 100 μM carbenoxolone (CBX) for 0.5, 3, 16, or 24 h. Briefly, 35 mm culture plants with MLO-Y4 were washed twice with DPBS. Subsequently, the cells were incubated in 0.2 mL DPBS solution containing the gap junction-permeable fluorescent dye, 1% Lucifer yellow and 1% of the gap junction-impermeable fluorescent dye, rhodamine dextran 10 kDa. Six scrapes, consisting of three horizontal and three vertical lines, were swiftly made with a scalpel across the cell culture plate. After 5 min of incubation, the dyes were washed four times with DPBS. The cells were then fixed with 2% PFA for 10 min at room temperature. Fluorescence images were captured using an inverted fluorescent microscope (BZ-X800 Keyence, Tokyo, Japan). The experiment was repeated three times, and data were quantified by measuring fluorescence areas of three fields using NIH ImageJ. Quantification of changes in gap junction coupling induced by different treatments was performed by measuring the fluorescence area in mm2 of the Lucifer yellow fluorescence minus rhodamine dextran fluorescence.

Transwell migration and invasion, and tumor spheroid formation assays

Transwell migration was performed following a previously described protocol.68 Breast cancer cell suspensions were added to the upper side of the chambers at a density of 4 × 104 cells/insert dissolved in CM, and 750 μL of CM was added to the lower wells. After incubation, the cells were fixed and stained with hematoxylin and eosin. The cells inside the insert were removed using clean foam swabs (Texwipe, Kernersville, NC, USA). The number of migrated cells in 5 fields of view per insert at 10× magnification was photographed and counted. Each experiment was repeated three times.

Tumor spheroid formation assay was performed following a previously described method.68 A total of 2,000 BT474 cells expressing GFP were seeded in round-bottom ultra-low attachment spheroid microplates on day 0 and cultured with the collected CM from osteocytes. Spheroid formation was observed 24 h after culturing initiation, and images were captured and tracked for the following seven days with a daily media change. The spheroid volume was calculated using ImageJ software (NIH), and live and dying cells within spheroids were labeled with Hoechst (1ug/ml, Invitrogen, MA, USA) and Propidium Iodide (10ug/ml, Invitrogen, MA, USA), respectively.

Conditional knockout mice

Mice with floxed Cx43 gene (Cx43flx/flx) were originally generated by Dr. Klaus Willecke’s lab at the University of Bonn, Germany64 and provided by Dr. Roberto Civitelli at Washington University. Mice with a deletion of Cx43 from osteocytes were generated using the Cre/Lox system. Initially, mice with the floxed Cx43 gene (Cx43flx/flx) were crossed with Cx43 heterozygous mice expressing one Cx43 allele (Cx43+/−). Subsequently, mice expressing a Cre recombinase driven by a 10-kb DMP1 promoter, which leads to gene expression predominantly in osteocytes65 (DMP1-Cre; Cx43+/+), were crossed with Cx43fl/− mice to generate Cx43 osteocyte-specific cKO (DMP1-Cre; Cx43fl/−) mice. Genotyping was performed using polymerase chain reaction (PCR) techniques with genomic DNA isolated from mouse tails and corresponding primers synthesized at the UTHSCSA DNA Core Facility.

Intratibial injection, mammary fat pad injection, intracardiac injection, bioluminescence imaging, and radiography

All mice were maintained in a pathogen free environment at the AAALAC-accredited UTHSCSA animal facility, following the NIH Guidelines for the Care and Use of Laboratory Animals. The animal experimental protocols complied with ethical regulations and were approved by the Institutional Animal Care and Use Committee (IACUC). Six to seven-week old female C57BL/6 mice were used for the intratibial injections as previously described.23 Animals were randomly divided into control and treated groups. Briefly, PY8119 and MDA-MB-231 cells expressing Luc-GFP (2×104 cells in PBS) were inoculated into the bone marrow of the left tibias. For Cx43-M2(MH) treated mice, the antibody (5–25 mg/kg) was i.p. injected once or twice a week. Intratibial tumor growth was monitored with bioluminescence imaging using the Xenogen IVIS-Spectrum imaging system (Alameda, CA, USA) every week starting from 3 days after tumor cell inoculation. Analysis was performed using LivingImage software (Xenogen) by measuring the photon flux (photons/sec/cm2/steradian) with a region of interest (ROI). Tumor burden was assessed by drawing an ROI around the major bioluminescence signal. Mice were also imaged with an X-ray at 35 KVP for 5 s using a Faxitron Digital Radiographic Inspection unit against the detector as described previously.69

Mouse plasma ATP quantification

WT mice were treated with 25 mg/kg of Cx43-M2(MH) antibody. After 0, 24, and 48 h, mice were anesthetized with Ketamine Xylazine. The blood sample was gently collected from the retroorbital artery (to avoid hemolysis) into an ice cooled tube with 60 U of heparin (#H3393, Sigma) and 10 μL of 20 mM ARL67156, and then centrifugated for 10 min at 0.5G at 4°C. The plasma was stored at −80°C. The ATP levels were measured by a luciferin/luciferase assay using the manufacturer’s protocol (ATP Determination Kit, #A22066 ThermoFisher).

Flow cytometry

Mouse samples were incubated with anti-CD16/32 (2.4G2) produced in the lab and used in all FACS staining as a Fc receptor blocker. Single cell suspension was surface-stained first on ice for 30 min with a cocktail of specific cell surface fluorescence dye-labeled antibodies against CD45.2, CD3, TCR-β, CD8b, CD4, CD25, and PD-1. Ghost Dye Violet 510 (Tonbo Bioscience) was used to identify live cells. For granzyme A and Foxp3 staining, surface-stained cells were treated by Foxp3/Transcription Factor Staining Buffer Kit (Tonbo). Washed and fixed samples were analyzed by BD LSRII or BD FACSCelesta and analyzed by FlowJo (TreeStar) software.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis

We performed preliminary experiments using 3–6 animals to calculate the total number of animals required to achieve 80% power, assuming two-sided testing with a significance level of 5%. Unless otherwise specified in Figure Legends, the data are presented as the mean ± SEM of at least three determinations. Asterisks indicate the level of significant differences compared to the controls (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Statistical parameters, including number of biological replicates, sample sizes, algorithm parameters are reported in the method details and the figure legends. One-way analysis of variance and the Student Newman-Keuls test were used to compare groups using GraphPad Prism 5.04 software (GraphPad, La Jolla, CA, USA).

Supplementary Material

1

ACKNOWLEDGMENTS

The authors would like to thank Hongyun Cheng for technical assistance, Dr. Lesley Ellies (then at the University of California, San Diego) for providing PY8119 cells, Dr. Lynda Bonewald (then at the University of Texas Health Sciences Center at San Antonio [UTHSCSA]) for the MLO-Y4 osteocytic cells, Dr. Stephen Harris at UTHSCSA for 10-kb dentin matrix protein 1 -Cre mice, and Dr. Klaus Willecke (then at the University of Bonn) and Dr. Roberto Civitelli at Washington University in St. Louis for mice with floxed Cx43 genes. The work was supported by Department of Defense BC161273 (to J.X.J. and Z.A.), NIH grant CA196214 (to J.X.J. and L.-Z.S.), Welch Foundation grants AQ-1507 (to J.X.J.) and AU-0042–20030616 (to Z.A.), the Cancer Prevention and Research Institute of Texas grants RP150551 and RP190561 (to Z.A.) and F32DK134051 (to F.M.A.), and the National Cancer Institute Cancer Center Grant 2 P30 CA054174–17 to the Cancer Therapy and Research Center.

Figure 1. The monoclonal Cx43-M2 antibody specifically targets Cx43

(A) Schematic illustrating the structure of connexin and interaction with the Cx43-M2 antibody. The Cx43-M2 antibody binds to the second extracellular domain.

(B) HeLa cells, with or without Cx43 transfection, were incubated with M2(MH) or M2(H) and detected using an ABC kit using peroxidase. Scale bar, 50 μm.

(C) Impermeable cells were immunostained with M2(MH) or M2(H) antibody (green) and counterstained with WGA (red), which detects glycoproteins on the cell surface. Scale bar, 50 μm.

(D) Kinetics of the M2(H) antibody binding to the Cx43 M2 peptide was assessed using an Octet RED96.

Figure 2. Cx43-M2 antibody activates hemichannels in osteocytes

(A) EtBr dye uptake was performed in MLO-Y4 cells with various doses of M2(MH).

(B) Gap junction coupling was determined using scrape wound dye transfer assay using Lucifer yellow (right images) in the absence and presence of CBX or M2(MH). Scale bar, 100 μm.

(C) Primary osteocytes were isolated from the femoral bone of 4-month-old mice, and EtBr dye uptake was performed in the absence or presence of CBX and M2(MH).

(D) EtBr dye uptake was performed in MLO-Y4 cells in the absence or presence of CBX and M2(H).

(E) CBX inhibits M2(MH)-induced release of ATP from MLO-Y4 osteocytes.

(F) MLO-Y4 osteocytes were treated with or without M2(MH), and CM was collected. MDA-MB-231 cells were incubated with the CM, and cancer cell migration was then analyzed using the transwell migration assay. Images of cells in the transwell assay are shown (left). Scale bar, 100 μm.

(G) MDA-MB-231 cells were treated with or without Cx43-M2(MH), and cell migration was analyzed using transwell.

All data are presented as means ± SEMs. n = 3–6. Data were analyzed using 1-way ANOVA for (A)–(D), 2-way ANOVA for (E), and the Student’s t test for (F) and (G). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. The Cx43-M2 antibody is delivered to osteocytes and activates Cx43 hemichannels in situ

(A) M2(MH) (25 mg/mL) was i.p. injected into the bone marrow of 4-month-old mice, and tibial cortical bone sections were prepared. Antibodies were detected using fluorescein isothiocyanate-conjugated anti-human secondary IgG (green) and counterstained with DAPI (blue). Scale bar, 50 μm.

(B) Evans blue dye was injected into the tail vein of 4-month-old mice 2 h after i.p. injection of M2(MH) or saline. Tibias were isolated, fixed, and demineralized 2 h after injection, and tibial tissue sections were prepared. Dye uptake was measured in the cortical and trabecular bone area by Evans blue fluorescence and counterstained with DAPI (blue) (lower left). Scale bar, 100 μm. The extent of dye uptake was quantified by NIH ImageJ software (right).

All data are presented as means ± SEMs from several images of 3 independent experiments. Data were analyzed using a Student’s t test. *p < 0.05; ***p < 0.001.

Figure 4. The Cx43-M2 antibody inhibits osteolytic breast cancer growth and preserves bone integrity

Luc-expressing breast cancer cells were injected into the bone marrow of the left tibias of female mice. The right tibias were injected with PBS as controls. M2(MH) was administrated via i.p. injection once or twice weekly for up to 4 weeks. (A and B) M2(MH) or human IgG (hIgG) at 25 mg/kg was i.p. injected once per week, and vehicle (saline) at the same volume was injected once per week in control mice.

(A) Representative bioluminescence images (top) and tumor tissues isolated (bottom) from the mice implanted with MDA-MB-231 cells for 4 weeks are presented after treatments.

(B) Tumor growth was recorded every week for 4 weeks by bioluminescence imaging and quantified. n = 6–8.

(C and D) M2(MH) at 25 mg/kg was i.p. injected once or twice per week. (C) Representative bioluminescence images of the mice implanted with PY8119 cells for 4 weeks are presented. (D) Tumor growth was recorded every week for 4 weeks. n = 3–4.

(E) M2(MH) at 5, 15, or 25 mg/kg was i.p. injected once per week. The tumor growth was recorded every week for 4 weeks by bioluminescence imaging and quantified. n = 6–15.

(F) M2(MH) at 25 mg/kg was i.p. injected once per week. Representative X-ray radiographs with the tibia injected with PY8119 cells at 4 weeks indicate where the tumor cells were implanted and osteolytic lesions occurred (dashed circles).

(G) M2(MH) at 25 mg/kg was i.p. injected once per week into WT and osteocyte-specific Cx43 cKO mice. Tumor growth in WT and cKO mice injected with M2(MH) or saline for 4 weeks was quantified; n = 3.

(H–J) Human MDA-MB-231-Luc (H), BT474-Luc (I), and T47D-Luc cells (J) were injected into the bone marrow of the right tibias of immune-compromised, athymic mice. The left tibias were injected with saline (controls). M2(H) at 5, 15, or 25 mg/kg, or saline was i.p. injected once per week, and tumor growth was recorded every week for 6 weeks by bioluminescence imaging and quantified. n = 6–8.

All data are presented as means ± SEMs. n = 3–8. Data were analyzed using 1-way ANOVA for (A)–(G) and 2-way ANOVA for (H)–(J). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. The Cx43-M2 antibody inhibits breast cancer migration via ATP release, increases circulating ATP, reduces breast cancer metastasis, and improves survival

(A and B) MLO-Y4 cells were treated with or without M2(MH), and CM was collected. MDA-MB-231 cells were incubated with different CM, and cancer cell migration was analyzed using the transwell migration assay. (A) MDA-MB-231 cells were treated with CM from control or M2(MH)-treated MLO-Y4 cells pretreated with or without ectoATPase inhibitor ARL-67156 before adding to MDA-MB-231 cells. n = 6. (B) MDA-MB-231 cells were treated with CM from control or M2(H)-treated MLO-Y4 cells pretreated with or without Apy before adding to MDA-MB-231 cells. n = 8.

(C and D) M2(MH) was administrated via i.p. injection once per week. Blood samples were collected, and the amount of ATP was analyzed using plasma at the post-M2(MH) treatment for 6, 24, and 48 h (C) or 24 h after treatment with M2(MH) at 5, 15, or 25 mg/kg (D). n = 3–5.

(E–G) M2(MH) reduces breast cancer metastasis and improves survival rate. Human MDA-MB-231-Luc (E and F) and murine PY8119-Luc cells (G and H) were injected into the heart cavity of athymic and C57BL/6 WT mice, respectively, and M2(MH) at 25 mg/kg was administrated once per week. Cancer metastasis was monitored by bioluminescence images. Representative bioluminescence images are presented after 4 weeks of treatment (E and G, left). The bioluminescence signals after 4 weeks of treatment were measured as total photon flux (E and G, center) and positive area (right). n = 15. Survival rates of the mice implanted with MDA-MB-231 cells (F) or PY8119 (H) were depicted using a Kaplan-Meier curve. MDA-MB-231 cells were concurrently implanted and administrated with M2(MH), while PY8119 cells were implanted 2 days after the first administration of M2(MH) (primed). n = 15.

All data are presented as means ± SEMs. Data were analyzed using 2-way ANOVA for (A) and (B), 1-way ANOVA for (C) and (D), a Student’s t test for (E) and (G), and the log rank Mantel-Cox test for (F) and (H). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6. The Cx43-M2 antibody increases cytotoxic and helper T-lymphocytes while suppressing regulatory T lymphocytes

PY8119-Luc cells were injected into the bone marrow of the right tibias of female WT mice. The left tibias were injected with PBS as controls. M2(MH) at a dosage of 25 mg/kg was administered via i.p. injection once per week for 4 weeks.

(A and B) T cells were isolated from the draining lymph nodes of WT mice with or without tumor implantation and treated with or without M2(MH) and subjected to separation via fluorescence-activated cell sorting (FACS) using anti-TCR-β/CD8 antibody for Tc, anti-TCR-β/CD4 antibody for Th, and CD3/CD4/CD25 for Tregs. Representative FACS profiles of pre-gated live lymphocytes isolated from the draining lymph nodes of tumor-bearing mice are shown in (A), and the percentages of positive cells out of GFP− (non-tumor cells) were quantified (B). n = 5–12.

(C) T cells were isolated from the blood of tumor-implanted mice treated with or without M2(MH) and subjected to separation via FACS as described in (B). n = 5–12.

(D) T cells were isolated from the implanted tumor treated with or without M2(MH) and subjected to separation via FACS for Tc, Th, Treg, and Gzma and PD-1+ CD8 cells. n = 4–5.

Data were analyzed by 1-way ANOVA for (B) and Student’s t test for (C) and (D). All data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7. Humanized Cx43-M2(H) inhibits the migration of human osteosarcoma cells via ATP and osteosarcoma growth in the bone

(A) CM from MLO-Y4 osteocytes treated with or without M2(H) was collected and treated with or without apyrase. Cell migration was analyzed in OS17 cells treated with the collected CM. Data points were normalized with respect to the average value of each control. Each value comes from 3 pictures from 3 independent experiments (n = 3).

(B) Athymic mice were intratibially injected with OS17 cells and treated with ATPgS at 25 mg/kg twice per week for 4 weeks, and bioluminescence signals were quantified. n = 4–6.

(C–F) Human osteosarcoma cell lines. OS17-Luc (C), 143B-Luc (D), and DLM-8-luc cells (E and F) were intratibially injected into the bone marrow of the right tibia of athymic mice, while the left tibias were injected with saline as controls. M2(H) for OS17 and 143B in athymic mice and M2(MH) in WT C56BL/6 mice were i.p. administered at 5, 15, and 25 mg/kg injected once per week. Tumor growth was assessed weekly using bioluminescence imaging and signals quantified with data collected after 42 days for OS17, 35 days for 143B, and 21 days for DLM-8 cells. (F) n = 6–8.

(G) Mice intratibially implanted with DLM8 cells were treated with 5, 15, and 25 mg/kg M2(MH), and survival rates were depicted using a Kaplan-Meier curve.

All data are presented as means ± SEMs. Data were analyzed using 1-way ANOVA for (A), (C)–(F), and Student’s t test for (B). *p < 0.05; **p < 0.01.

KEY RESOURCES TABLE REAGENT or RESOURCE	SOURCE	IDENTIFIER	
	
Antibodies	
	
Cx43-M2	This paper	N/A	
Cx43-M2(MH)	This paper	N/A	
IgG from human serum	Sigma-Aldrich	Cat# 14506; RRID: AB_2827935	
mouse-IgG-control-human	Santa Cruz Biotechnology	Cat# sc-2025; RRID: AB_737182	
PE/Cyanine7-anti-PD-1 (29F.1A12)	Biolegend	Cat# 135216; RRID: AB_10689635	
eFIuorTM 450 anti-CD8b (H35–17.2)	Thermo Fisher Scientific	Cat# 48008382; RRID: AB_11218504	
Brilliant Violet 785 anti-CD45.2 (104)	BioLegend	Cat# 109839; RRID: AB_2562604	
APC-Cy7-anti-TCR-β (H57–597)	BioLegend	Cat# 109220; RRID: AB_893624	
PE-anti-CD25 (PC61)	Biolegend	Cat# 102008; RRID: AB_312857	
Alexa Fluor 488-anti-Foxp3 (MF-14)	Biolegend	Cat# 135216; RRID: AB_10689635	
PerCP-Cy5.5-anti-CD4 (GK1.5)	Biolegend	Cat# 100434; RRID: AB_893324	
Anti-CD16/32 (2.4G2)	Produced in-house	N/A	
APC anti-Granzyme A (GzA-3G8.5)	Thermo Fisher Scientific	Cat# 17583182; RRID: AB_2573228	
Alexa Fluor® 488 anti-human IgG Fc Antibody	Thermo Fisher Scientific	Cat# SA510134; RRID: AB_2556714	
Mouse Anti-Human IgG4 Fc-Alexa Fluor® 488	SouthernBiotech	Cat# 9200–30; RRID: AB_2796694	
	
Bacterial and virus strains	
	
pIRES2-EGFP-Cx43 WT	Dr. Bruce Nicholson	N/A	
	
Chemicals, peptides, and recombinant proteins	
	
Wheat Germ Agglutinin	Invitrogen	Cat# W11261	
M2 peptide	This paper	N/A	
Carbenoxolone	Thermo Fisher Scientific	Cat# 501786420	
Lucifer Yellow CH, Lithium Salt	Invitrogen	Cat# L453	
Ethidium Bromide	Amresco	Cat# 0492	
Apyrase	Thermo Fisher Scientific	Cat# A6237	
ARL 67156 Trisodium Salt	Thermo Fisher Scientific	Cat# 12–831-0	
Adenosine Triphosphate	Sigma-Aldrich	Cat# A26209	
DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride)	Thermo Fisher Scientific	Cat# PI62247	
Evans Blue	Thermo Fisher Scientific	Cat# 195550250	
Luciferase from Photinus Pyralis	Sigma-Aldrich	Cat# L9420	
Type 1 Collagenase	Worthington Biochemical Corporation	Cat# 354236	
Ghost DyeTM Violet 510	Cytek (Tonbo)	Cat#13–0870-T500	
Fetal bovine serum	Corning	Cat# 35–015-CV	
Pannexin-1 (Panx1), Mimetic Blocking Peptide	AnaSpec	Cat# 61911	
Penicillin-Streptomycin	Corning	Cat# 30001CI	
Bovine calf serum	Cytiva HyClone	Cat# SH30073.03H	
Perkin Elmer LLC XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate	Thermo Fisher Scientific	Cat# 122799	
ATPƔS	Thermo Fisher Scientific	Cat# 40–801-0	
Calcein, AM	Invitrogen	Cat# C3100MP	
Dil Stain (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate (‘DiI’; DiIC18(3)))	Invitrogen	Cat# D3911	
Propidium Iodide	Thermo Fisher Scientific	Cat# 50–182-62	
Hoechst	Thermo Fisher Scientific	Cat# H21492	
BzATP	Sigma Aldrich	Cat# B6396	
Autocamtide-2-related Inhibitory Peptide	AnaSpec	Cat# AS-64929	
Oxidized ATP	Thermo Fisher Scientific	Cat# 50–575-80001	
Brilliant Blue G	Brilliant Blue G	Cat# 6104–58-1	
A804598	Thermo Fisher Scientific	Cat# 44–731-0	
	
Critical commercial assays	
	
ATP Determination Kit	Thermo Fisher Scientific	Cat# A22066	
Hema3 Stat Pack	Thermo Fisher Scientific	Cat# 23–123869	
Corning 8μm Transwell Inserts	Thermo Fisher Scientific	Cat# 07–200-150	
VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Rabbit IgG)	Vector Laboratories	Cat# 32054	
Foxp3/Transcription factor staining buffer kit	Cytek (Tonbo)	Cat#TNB-0607	
	
Experimental models: Cell lines	
	
Mouse: MLO-Y4 cells	Dr. Lynda Bonewald	RRID: CVCL_M098	
Human: MDA-MB-231-Luc cells	Dr. Luzhe Sun	RRID: CVCL_C8W9	
Mouse: Py8119-Luc cells	Dr. Luzhe Sun	RRID: CVCL_AQ09	
Human: HeLa cells	Dr. Luzhe Sun	RRID: CVCL_0030	
Human: BT474-Luc cells	Dr. Luzhe Sun	RRID: CVCL_VL01	
Human: T47D-Luc cells	Dr. Luzhe Sun	RRID: CVCL_0I95	
Human: OS17-Luc cells	Dr. Peter Houghton	RRID: CVCL_Z613	
Human: 143B-Luc cells	ATCC	Cat# CRL-8303; RRID: CVCL_2270	
Mouse: DLM8-Luc cells	Dr. Luzhe Sun	RRID: CVCL_6669	
	
Experimental models: Organisms/strains	
	
Mouse: Cx43 cKO (DMP1-Cre; Cx43flx/flx)	Dr. Stephen Harris; Dr. Klaus Willecke	Strain: 129P2/OlaHsd	
Mouse: Athymic nude mice	ENVIGO	Hsd: Athymic Nude-Foxn1nu	
	
Software and algorithms	
	
ImageJ	National Institutes of Health	https://imagej.nih.gov/ij/	
GraphPad Prism 8	GraphPad, Inc	https://www.graphpad.com/scientific-software/prism/	
Octet Red96	FortéBio	https://www.creative-biolabs.com/Octet-system.html	
FlowJo v10	Treestar Inc	RRID: SCR_008520	
	
Other	
	
The Cancer Genome Atlas (TCGA)	National Cancer Institute	http://cancergenome.nih.gov/	
Microscope Olympus	https://www.olympus-ims.com/en/user-manuals/	IX70	
Microscope Keyence	Keyence, Inc	BZ-X710	
Faxitron X-ray	Faxitron X-ray, Inc	Model LX-60	

Highlights

Developed Cx43-M2 antibody that activates Cx43 hemichannels in osteocytes

Cx43-M2 reduces breast cancer and osteosarcoma cell growth and improves survival rates

Cx43-M2 enhances anti-tumor immunity by activating Tc cells while suppressing Treg cells

ATP release by hemichannels and purinergic signaling inhibits tumor and enhances immunity

DECLARATION OF INTERESTS

J.X.J. is a co-founder, shareholder, and a member of the Scientific Advisory Board of AlaMab Therapeutics. J.X.J., M.A.R., S.G., W.X., N.Z., and Z.A. are listed as inventors on relevant patent applications that were exclusively licensed to AlaMab Therapeutics by the Board of Regents of the University of Texas system. The University of Texas has a financial interest in AlaMab in the form of licensing.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114377.
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