
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39251652
71140
10.1038/s41598-024-71140-9
Article
Veillonella parvula as an anaerobic lactate-fermenting bacterium for inhibition of tumor growth and metastasis through tumor-specific colonization and decrease of tumor’s lactate level
Kefayat Amirhosein 1
Bahrami Mahshid 2
Karami Mojtaba 3
Rostami Soodabeh 4
Ghahremani Fatemeh bahare.ghahremani@gmail.com
f.ghahramani@arakmu.ac.ir

5
1 https://ror.org/04waqzz56 grid.411036.1 0000 0001 1498 685X Department of Oncology, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
2 https://ror.org/04waqzz56 grid.411036.1 0000 0001 1498 685X Department of Radiology, Isfahan University of Medical Sciences, Isfahan, Iran
3 https://ror.org/01c4pz451 grid.411705.6 0000 0001 0166 0922 Department of Dermatology, Tehran University of Medical Sciences, Tehran, Iran
4 https://ror.org/04waqzz56 grid.411036.1 0000 0001 1498 685X Nosocomial Infection Research Center, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
5 https://ror.org/056mgfb42 grid.468130.8 0000 0001 1218 604X Department of Medical Physics and Radiotherapy, Arak University of Medical Sciences, Sardasht, Meydan Basij, Arāk, 38481-76941 Iran
9 9 2024
9 9 2024
2024
14 210084 2 2024
26 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
High tumor’s lactate level directly associates with high tumor growth, metastasis, and patients’ poor prognosis. Therefore, many studies have focused on the decrease of tumor’s lactate as a novel cancer treatment. In the present study for the first time, a strictly anaerobic lactate-fermenting bacterium, Veillonella parvula, was employed for the decrease of tumor’s lactate level. At first, 4T1 breast tumor-bearing BALB/c mice were administered with 106 V. parvula bacteria intravenously, orally, intraperitoneally, and intratumorally. Then, the bacteria biodistribution was evaluated. The best administration route according to tumor colonization was selected and its safety was assessed. Then, the therapeutic effect of V. parvula administration through the best route was investigated according to 4T1 murine breast tumor’s growth and metastasis in vivo. In addition, histopathological and immunohistochemistry evaluations were done to estimate microscopic changes at the inner of the tumor and tumor’s lactate level was measured after V. parvula administration. V. parvula exhibited considerable tumor-targeting and colonization efficacy, 24 h after intravenous administration. Normal organs were free of the bacteria after 72 h and no side effect was observed. Tumor colonization by V. parvula significantly decreased the tumors’ lactate level for about 46% in comparison with control tumors which caused 44.3% and 51.6% decline (P < 0.05) in the mean tumors’ volume and liver metastasis of the treatment group in comparison with the control group, respectively. The treatment group exhibited 35% inhibition in the cancer cell proliferation in comparison with the control according to the Ki-67 immunohistochemistry staining. Therefore, intravenous administration of V. parvula is a tumor-specific and safe treatment which can significantly inhibit tumors’ growth and metastasis by decreasing the tumor lactate level.

Keywords

Breast cancer
Bacteria therapy
Veillonella parvula
Lactate
Subject terms

Bacteria
Cancer
Microbiology
Oncology
http://dx.doi.org/10.13039/501100007113 Arak University of Medical Sciences 67354 Ghahremani Fatemeh issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Breast cancer is the leading cause of cancer-related deaths among women, worldwide1. Although breast cancer treatments have achieved considerable improvements in recent years, patients’ prognosis is not satisfying yet, especially for metastatic breast cancer. Metastasis is the most pernicious behavior of cancer cells and it is responsible for more than 90% of cancer patients’ death2. Neoplasms arise from malignant transformations of normal cells which cause gaining specific features and abilities like limitless growth and proliferation, avoidance of apoptosis, sustained angiogenesis, metastasis, and specific metabolic pathways in the cancer cells3.

Uncontrolled growth and proliferation of cancer cells is highly energy consuming and need biomaterials4. To carry out these needs, malignant cells require redesigning of their metabolic pathways in comparison with the normal cells and give rise to their specific metabolism5. A tumor can be divided into two regions according to the oxygen concentration, including well-oxygenated (aerobic) and poorly oxygenated (hypoxic) regions. Inhabitants of these two regions initiate a metabolic symbiosis due to resources limitations6–8. Therefore, hypoxic regions cells enhance glucose intake and due to the enhanced glycolytic carbon flux, hypoxic cells exhibit high lactate production. To prevent intracellular acidification, hypoxic cells secret lactate to the extracellular fluid. On the other hand, aerobic cells at normoxic regions of tumor microenvironment internalize the secreted lactate instead of glucose to complete the intratumoral metabolic symbiont cycle. The internalized lactate will convert to pyruvate to produce the Krebs cycle fuel for energy production9–12. Many studies have targeted decrease of lactate in the tumor microenvironment as a new therapeutic method for cancer13–15. These agents cause disruption of the metabolic symbiosis in tumor and subsequent cancer cells’ death in the hypoxic zone8,9. This phenomenon has gained lots of attention due to the determinative role of tumor hypoxic regions for resistance to current cancer treatments16–18. High lactate production at the tumor causes formations of an acidic microenvironment which is associated with metastasis, angiogenesis and therapy resistance, a characteristic phenotype of more aggressive tumors. High lactate level in the tumor microenvironment promotes immunosuppression thus preventing the recognition of tumor cells by the immune system and inhibits lymphocytes activity. Also, many studies have reported the direct relation of the tumor’s lactate level with developing metastasis and cancer patients’ poor prognosis and introduced intratumor lactate level as a prognostic and response to treatment-monitoring biomarker19–23.

Recently, employing bacteria for cancer therapy has gained lots of attention. Different bacteria species have been utilized for tumor drug delivery24, anti-tumor immune therapy25, tumor colonization and direct invasion to the cancer cells26 which can cause tumor growth retardation and even complete tumor eradication27,28. In addition, bacteria can create anti-tumor effects through the depletion of nutrients and resources competition with cancer cells29. Moreover, anaerobic bacteria have exhibited high efficacy for cancer treatment due to selective tumor targeting and colonization at hypoxic regions30,31. William B. Coley was the pioneer in employing bacteria for cancer therapy. He utilized alive Streptococcus pyogenes for treatment of a head and neck cancer patient which caused hopeful results32. Subsequently, over one thousand cancer patients were treated with Coley’s toxins and excellent results were observed in a 40 years period33,34. Rise of radiation therapy at that time caused the fading of the bacteria therapy outcomes. But, the resistance of cancer cells to the current treatments and extensive side effects of them made researchers focus on the new or even forgotten therapeutic approaches like bacteria therapy for cancer treatment27,35,36.

The tumor is a hospitable microenvironment for the bacteria localization and colonization. Bacteria can hide from the immune system at the tumor site37 and the tumor microenvironment can provide enough nutrition for them29. Tumor hypoxia is one of the main predisposing factors for tumor colonization of bacteria38,39. Therefore, anaerobic species like Escherichia40, Clostridium41, Bifidobacterium42,43, Salmonella44, and Listeria45 have gained lots of attention for cancer treatment due to tumor-specific colonization after systemic administration. Also, tumor hypoxic regions are the main site of chemo and radiation therapy resistance. These regions have a determinative effect on the patients’ prognosis46. Bacteria colonization at the tumor site can cause anti-tumor effects through different mechanisms including activating the immune system and recruitment of immune cells to the tumor47,48, production of anti-tumor substances27, induction of cancer cells apoptosis due to intracellular proliferation, and competition for nutrition resources49.

In the present study, we hypothesized to treat murine breast tumors by employing an anaerobic bacterium with a high preference for lactate consumption to disrupt tumor metabolism. Veillonella parvula is a strictly anaerobic lactate-fermenting species which is highly vulnerable against oxygen50. This feature can limit its localization and colonization just at the tumor site38. Also, V. Parvula is not a pathogen and lives in human and animals’ gastrointestinal tract. V. parvula uptakes lactate as a determinative energy resource for cancer cells and mainly produces propionate51 which have anti-tumor effects52,53. According to the best of our knowledge, this is the first time to use a bacterium to interrupt tumor lactate metabolism for inhibiting tumor’s growth.

Method and materials

Bacterium culture

V. parvula (ATCC 10790) was cultured on Reinforced Clostridial medium (Oxoid CM149) with sodium lactate (60% solution) at a concentration of 1.5% at 37 °C in the anaerobic atmosphere (gas mixture, 80% N2-10% CO2-10% H2) for 5 days54.

Cancer cells culture

Mouse mammary carcinoma cell line (4T1) was purchased from Pasteur Institute of Tehran, Iran. Cells were cultured in RPMI 1640 medium (Sigma, USA) containing 10% fetal bovine serum (FBS) (Sigma, USA) and 1% antibiotics mixture containing penicillin (Sigma, USA) and streptomycin (Sigma, USA). The cells were incubated at 37 °C in a humidified incubator at 5% CO2 atmosphere54.

Animals’ husbandry and handling

Female BALB/c mice (age, 6–8 weeks; mean weight, 25 ± 2 g) were purchased from the Pasteur Institute of Tehran, Iran. The mice were maintained at 24 ± 2 °C temperature, 50 ± 10% relative humidity, and 12 h light/12 h dark cycle condition with complete access to standard mouse chow and water. The mice were acclimated for at least 1 week before the start of the study. If any signs of pain, wounds, massive necrosis and hemorrhage, diffuse metastasis (observation of abdominal swelling or breathing difficulties) were observed during any steps of the study, the mice were sacrificed by Ketamine/Xylazine solution overdose54.

4T1 cancer cells implantation

BALB/c mice were injected subcutaneously (s.c) into the 4th abdominal mammary fat pad with 1 × 106 4T1 cells suspended in 50 µl PBS. The injection site was shaved and sterilized by 70% alcohol before injection. In order to determine tumors’ growth progression, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) of the tumors were determined every 3 days for 22 days after the cancer cells implantation. Then, the tumor’s volume was calculated by the tumor volume Eq. (1)55. One group of mice were injected with PBS as the no-treatment group (control).1 Tumorvolume=tumorlength×tumorwidth2/2

Bacteria biodistribution

To evaluate the bacteria biodistribution in tumor-bearing mice body, three tumor-bearing mice which were i.v injected with 106 V. parvula were sacrificed after 24 and 72 h. The tissue samples from the tumor, liver, lung, blood, spleen, and kidney were prepared. The tissues were weighed, and then 0.1 g of each tissue were homogenized in 3 ml of PBS at room temperature for 1 min in a completely aseptic condition to extract the bacteria from tissues. Then, to determine the presence of the bacteria, 10 µl homogenized tissues were plated onto the standard culture medium for V. parvula and incubated 72 h in anaerobic condition and bacterial colony count was evaluated in colony forming units (CFUs)54.

Histopathology and immunohistochemistry exams

The mice were sacrificed by over-dose of Ketamine/Xylazine and their tumors were harvested. The tumors were fixed in 10% formalin neutral buffer solution and the fixed specimens were processed overnight for dehydration, clearing, and impregnation using an automatic tissue processor (Sakura, Japan). Then, the specimens were embedded in paraffin blocks and serial sections of 4 µm thickness were cut using a microtome (Leica Biosystems, Germany). The sections were stained by Hematoxylin & Eosin (H&E)56. The histopathology of blindly labeled slides was reviewed independently by two expert pathologists under a dual-head light microscope (Olympus, Japan). Both the low-power and high-power fields were examined. 5 µm sectioned were prepared from tissues blocks and stained by hematoxylin and eosin. Also, some tissue sections were stained with gram staining protocol according to previous studies57,58. Some tumor sections were stained for Ki-67 biomarker by immunohistochemistry according to our previous studies59. Histological photographs were captured using a digital light microscope (Olympus, Japan).

Tumor lactate assay

Tumor-bearing mice (n = 3) were i.v injected with 106 V. parvula at the 6th day after cancer cell implantation. The mice were sacrificed after 4 days and the tumor tissues were harvested and frozen in liquid nitrogen. Then, the tumor tissue samples were homogenized on ice in 4 volumes of lactate assay buffer and subsequently centrifuged at 13,000 for 10 min to remove insoluble material. 10 kDa spin filter was utilized to deproteinize the samples and removing endogenous lactate dehydrogenase. The soluble fraction was then assayed by Lactate assay kit (Sigma, USA) according to the manufacturer protocol.

Statistical analyses

The statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test by JMP 11.0 software (SAS Institute, Japan). Statistical significance was set at P < 0.05 (*P < 0.05, ns: not significant). The results were illustrated as mean ± standard deviation (SD). All experiments were replicated at least three times54.

Results

Biodistribution, tumor targeting, colonization, and safety efficacy of the V. parvula after systemic administration

The main determinative factor for the efficacy of a bacterium species for cancer treatment is the tumor targeting and colonization efficacy. It is obvious that more tumor colonization and less preference for the normal organs localization can significantly enhance the therapeutic efficacy and decrease the side effects60. Also, the administration route can significantly affect bacteria therapy outcome. Four common routes including intraperitoneal (i.p), intravenous (i.v), oral, and intratumoral (i.t) injections have been utilized for bacteria therapy in cancer61. Therefore, 106 V. parvula was injected i.p, i.v, oral, and i.t to the 4T1 breast tumor-bearing BALB/c mice to identify the best route of V. parvula administration. Tumors had 100–150 mm3 volume at the injection day. Tissue samples from the tumor, liver, lung, spleen, kidney, and blood of the tumor-bearing mice were collected and biodistribution of the bacteria was evaluated 24 h and 72 h after injection (Fig. 1). I.v administration of V. parvula caused the highest efficacy for tumor colonization in comparison with other administration routes (Fig. 1A). The bacteria were eradicated from blood circulation in less than 24 h after injection as no colony formation was observed. Therefore, the increase in the number of bacteria in the tumor site from 24 to 72 h since administration can be attributed to bacterial propagation within tumor. Although extremely limited V. parvula localization at the normal organs after administration was detected with first 24 h, it was temporary and the bacteria were eradicated from these organs after 72 h (Fig. 1B) and the bacteria just remained at the tumor site. During intertumoral injection of the bacteria solution, most of the solution escaped from the (subcutaneously implanted) tumors and entered the surrounding subcutaneous space which caused swelling during injection due to subcutaneous collection of the solution. The subcutaneous fluid was reabsorbed in less than 1 h. Therefore, low efficacy of intratumoral injection can be attributed to the low compliance of these tumor for receiving intratumoral injection solution due to their solid texture. On the other hand, intravenously injected bacteria can diffusely reach to the different parts of tumor through its dense, tortuous, and hyperpermeable vasculature. The main route of entering to the blood circulation after intraperitoneal injection, is lymphatic system drainage. However, it seems most of the administered bacteria trapped in the peritoneal lymphatic drainage system system after intraperitoneal injection, which is part of the immune system.Fig. 1 Efficacy of different routes of V. parvula systemic administration according to the bacteria biodistribution and tumor colonization at the 4T1 breast tumor-bearing mice after (A) 24 h (n = 3) and (B) 72 h (n = 3) since the injection (*P˂0.05).

Safety is one of the main concerns of utilizing bacteria for cancer treatment due to the colonization of normal tissues and systemic infection27. Therefore, the safety of i.v administration of V. parvula as the most effective administration route (according to tumor colonization parameter) was evaluated by blood biochemistry and histopathological assays. Healthy BALB/c mice (n = 5) were i.v injected with 1 × 106 and sacrificed after 30 days. Macroscopic examination of all the vital organs including liver, kidneys, lungs, and brain did not show any changes as compared to the control mice organs. Furthermore, plasma levels of AST and ALT were measured for liver function analysis. Increase in these enzymes’ plasma levels shows liver damage62. As Fig. 2A illustrates, both of these biomarkers’ levels were in the normal range as compared to the control group. This fact was the same for creatinine as a well-known kidney function biomarker63 (Fig. 2A). Besides, no sign of inflammation, abscess lesion formation, parenchymal architecture change, or fibrosis was observed in the vital organs according to histopathological analyzes (Fig. 2B). Taking together, V. parvula has high ability for tumor-specific targeting, localization, and colonization. It can significantly colonize at the tumor after i.v administration, with no significant side effects on the normal organs. Also, V. parvula colonization at the tumor site was evaluated weekly for 4 weeks after i.v administration. The bacteria remained detectable (CFUs > 106) at the tumor tissue within these 4 weeks after iv injection of the bacteria (Fig. S1).Fig. 2 (A) Blood biochemical analysis including AST, ALT, and creatinine and (B) histopathological analyzes of the non-tumor bearing (healthy) mice (n = 5), 30 days after i.v administration of 106 V. parvula (treatment) in comparison with the control.

Macroscopic and microscopic effects of V. parvula colonization at the breast tumors

4T1 breast tumor-bearing mice were i.v injected with 106 V. parvula. To investigate the macroscopic and microscopic effects of V. Parvula colonization at the breast tumors, the tumor-bearing mice (n = 5) were sacrificed 3 days after i.v administration of 106 V. parvula. As Fig. 3A illustrates, the tumors at the V. parvula-treated group exhibited pus formation which contained a high concentration of alive V. parvula according to gram staining and culturing the pus samples (data not-shown). Also, tumor H&E-stained sections exhibited clustered colonies of V. parvula at the central regions of the tumor (Fig. 3B,C) which is mostly hypoxic and necrotic regions. The peripheral regions were free of any colonies of bacteria (Fig. 3D). Taking together, multiple colonies of V. parvula were observed at the central regions of the breast tumors which demonstrates the high efficacy of this anaerobic species to colonize at the central regions of solid tumors while peripheral regions are free of V. parvula.Fig. 3 4T1 breast tumor colonization by V. parvula after i.v administration. (A) Macroscopic view of 4T1 tumors in the control and V. parvula administered tumor-bearing mice (72 h after i.v administration of 106 V. parvula, n = 5, mean tumors’ volume: 368 ± 80 mm3). Some of the tumors exhibited pus formation (Yellow arrow: pus, black arrow: tumor). (B) Low (× 4) and (C) high-power (× 100) field microscopic view of the H&E-stained sections of 4T1 breast tumors of the control and V. parvula administered groups (N: necrosis, V: viable, black arrows indicate the clustered colonies of V. parvula). The boxed regions in the panel B are viewed under high power in the panel (C). (D) High-power (× 400) field microscopic view of the central and peripheral regions of the H&E-stained sections of 4T1 breast tumors of V. parvula administered groups. The colonies of V. parvula were just observed at the central regions of the tumors and peripheral regions were bacteria free.

Effect of V. parvula colonization on the tumors’ growth and metastasis

4T1 breast tumor-bearing mice were i.v injected with 106 V. parvula on the 9th day after the cancer cells implantation. The mean tumor volume was 109 ± 18.6 mm3. The V. Parvula colonization at the tumor site caused significant inhibition (P < 0.05) of the tumor growth progression at the first days after administration in comparison with the control. On the last days of monitoring tumor growth, 44.3% decrease in the mean tumors volume of the treatment group in comparison with the control group was observed (P < 0.05) (Fig. 4A). Moreover, the treatment group exhibited significantly lower metastatic colonies (51.6%, Fig. 4B) at the H&E-stained sections of livers in comparison with the control group (Fig. 4C). In addition, to investigate the effect of V.parvula presence in the tumor microenvironment on the cancer cell proliferation, tumor sections were stained for Ki-67 marker by immunohistochemistry (Fig. 4D). As Ki-67-positive cells are the cells at the proliferation phase, this marker exhibits proliferation rate at the tumor. About 35% decrease in the Ki-67-positive cell (proliferation rate) was observed after treatment by V. parvula64.Fig. 4 The therapeutic effects of i.v administration of V. parvula on the 4T1 breast tumors’ growth progression, metastasis, and proliferation rate. 4T1 tumor-bearing mice were i.v injected with 106 V. parvula on the 9th day after cancer cells implantation (Mean tumors volume: 109 ± 18.6 mm3). The black arrow indicates the V. parvula injection day. (A) The mean tumors’ volume progression of the control and V. parvula groups (*P < 0.05, ns: not significant). (B) The average number of metastatic colonies at 10 random high-power microscopic fields of the H&E-stained sections of the liver at the 40th day after cancer cell implantation (n = 5). (C) The high-power microscopic (× 200) field of the H&E-stained sections of liver from the control and V. parvula administered groups. The red arrows point to some of the metastatic colonies as sample. (D) The high-power microscopic (× 400) field of the immunohistochemistry Ki-67-stained sections of tumor from the control and V. parvula-treated groups. The black arrow indicates the Ki-67-positive cells as a sample.

Effect of V. parvula colonization on the breast tumor’s lactate level

V. parvula is a lactate-fermenting bacterium which can considerably colonize the tumor. Therefore, its colonization at the tumor site may affect the tumor’s lactate level. Lactate level of the tumors is directly associated with their growth, invasion, metastasis, and cancer patients’ poor prognosis. Therefore, the decrease of the tumor lactate level can cause considerable therapeutic effects19,65,66. The treatment group was i.v injected with 106 V. parvula. The tumors’ lactate level was compared between the control and treatment groups on the 10th day after the cancer cells implantation. According to previous studies, 4T1 tumors exhibit the highest lactate level on the 10th day after the cancer cells implantation67. Lactate level of tumors in the treatment group was significantly (P < 0.05) lower than the control (Fig. 5). Therefore, V. parvula colonization at the tumor can insert its therapeutic effect by decrease of the tumors’ lactate level.Fig. 5 Assessment of lactate level in the 4T1 breast tumors after the bacteria administration (n = 3) (*P < 0.05).

Discussion

Many different bacteria species have exhibited high ability for colonizing solid tumors which surprisingly caused anti-tumor effects. Two of the most utilized bacteria for cancer treatment are Salmonella and Clostridium. Both are pathogen species which can kill the host if left untreated. Therefore, their attenuated subtypes were designed by genetic manipulation for in vivo experiments33,68. In the present study, V. parvula was employed for treatment of 4T1 breast tumors treatment in BALB/c mice. Extremely rare case studies have reported the V. parvula infection69,70. Intravenous administration of this bacteria was completely safe with high tumor-specific colonization. In addition, the unwanted localization of the bacteria at the normal organs was eradicated after 72 h and the tumor was the only niche for the bacteria localization and colonization which can be attributed to strictly anaerobic nature of this bacteria. Facultative anaerobic bacteria like Salmonella are not limited to the hypoxia and can colonize normal tissues which are a negative point for utilizing this species for tumor treatment30. V. parvula is a lactate-fermenting bacterium and its colonization at 4T1 tumors could significantly (P < 0.05) decrease the tumor lactate level. Indeed, lactate is the main fuel for cancer cells metastasis and have a direct relation with tumor recurrence and cancer patients’ poor prognosis66,71. V. parvula consumes lactate at the tumor and the main product of its metabolism is propionate51 which has proliferation-inhibiting, invasion-inhibiting, and apoptosis-promoting effects on the cancer cells52,53. All these observations can explain why significant tumor growth inhibition was observed at the V. parvula treated mice. Therefore, V. parvula has the potential to be an anti-tumor agent for breast tumor treatment. This bacterium never has been reported as part of tumor microbiome so far72. While, a recent study reported association of V. parvula decrease in saliva microbiome and oral squamous cell carcinoma.

Conclusions

Bacteria therapy has gained lots of attention for cancer treatment. In the present study, V. parvula as an anaerobic lactate-fermenting bacterium was administered through different routes to 4T1 breast tumor-bearing mice. The best administration route for V. parvula was intravenous which V. parvula could significantly colonize at the tumor and the localized bacteria at the normal organs were eradicated after 72 h. V. parvula colonization at the 4T1 breast tumor inhibited their growth progression and metastasis. One of the reasons for this therapeutic effect can be decreased tumor lactate level.

Supplementary Information

Supplementary Information.

Abbreviations

V. parvula Veillonella parvula

i.v Intravenous

i.p Intraperitoneal

i.t Intratumoral

CFUs Colony forming units

H&E Hematoxylin and eosin

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71140-9.

Acknowledgements

We would like to appreciate Arak University of Medical Sciences and Isfahan University of Medical Sciences.

Author contributions

A.K., S.R., and F.G. conceived and designed the experiment. The bacteria culture and preparation were performed by S.R. All the animal experiment, data analyses, and manuscript writing were performed by A.K., F.G., M.B., M.K., S.R.

Funding

This study was funded by the Ministry of Health and Medical Education of Iran and supported by Arak University of Medical Sciences (Grant Number: 3343).

Data availability

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare no competing interests.

Ethics statement

All experiments were done according to the Guidelines for the Care and Use of Laboratory Animals of Arak University of Medical Sciences, which refer to American Association for Laboratory Animals Science and the guidelines laid down by the NIH (NIH Guide for the Care and Use of Laboratory Animals) in the USA. All experimental protocols were approved by the animal research ethics committee of Arak University of Medical Sciences, Iran (IR.ARAKMU.REC.1398.012). All methods were performed in accordance with ARRIVE guidelines.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Luo C Global and regional trends in incidence and mortality of female breast cancer and associated factors at national level in 2000 to 2019 Chin. Med. J. 2022 135 01 42 51 10.1097/CM9.0000000000001814 34593698
Luo, C. et al. Global and regional trends in incidence and mortality of female breast cancer and associated factors at national level in 2000 to 2019. Chin. Med. J. 135(01), 42–51 (2022).34593698 10.1097/CM9.0000000000001814
2. Ganesh K Massagué J Targeting metastatic cancer Nat. Med. 2021 27 1 34 44 10.1038/s41591-020-01195-4 33442008
Ganesh, K. & Massagué, J. Targeting metastatic cancer. Nat. Med. 27(1), 34–44 (2021).33442008 10.1038/s41591-020-01195-4
3. Bisoyi, P. A brief tour guide to cancer disease. In Understanding Cancer 1–20 (Elsevier, 2022).
4. Schiliro C Firestein BL Mechanisms of metabolic reprogramming in cancer cells supporting enhanced growth and proliferation Cells 2021 10 5 1056 10.3390/cells10051056 33946927
Schiliro, C. & Firestein, B. L. Mechanisms of metabolic reprogramming in cancer cells supporting enhanced growth and proliferation. Cells 10(5), 1056 (2021).33946927 10.3390/cells10051056
5. Kroemer G Pouyssegur J Tumor cell metabolism: Cancer’s Achilles’ heel Cancer Cell 2008 13 6 472 482 10.1016/j.ccr.2008.05.005 18538731
Kroemer, G. & Pouyssegur, J. Tumor cell metabolism: Cancer’s Achilles’ heel. Cancer Cell 13(6), 472–482 (2008).18538731 10.1016/j.ccr.2008.05.005
6. Wilde, L., et al. Metabolic coupling and the reverse Warburg effect in cancer, implications for novel biomarker and anticancer agent development. In Seminars in Oncology (Elsevier, 2017).
7. Pavlides S The reverse Warburg effect: Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma Cell Cycle 2009 8 23 3984 4001 10.4161/cc.8.23.10238 19923890
Pavlides, S. et al. The reverse Warburg effect: Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle 8(23), 3984–4001 (2009).19923890 10.4161/cc.8.23.10238
8. Semenza GL Tumor metabolism: Cancer cells give and take lactate J. Clin. Investig. 2008 118 12 3835 3837 19033652
Semenza, G. L. Tumor metabolism: Cancer cells give and take lactate. J. Clin. Investig. 118(12), 3835–3837 (2008).19033652
9. Sonveaux P Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice J. Clin. Investig. 2008 118 12 3930 3942 19033663
Sonveaux, P. et al. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J. Clin. Investig. 118(12), 3930–3942 (2008).19033663
10. Romero-Garcia S Tumor cell metabolism: An integral view Cancer Biol. Therapy 2011 12 11 939 948 10.4161/cbt.12.11.18140
Romero-Garcia, S. et al. Tumor cell metabolism: An integral view. Cancer Biol. Therapy 12(11), 939–948 (2011).10.4161/cbt.12.11.18140
11. Fu Y The reverse Warburg effect is likely to be an Achilles’ heel of cancer that can be exploited for cancer therapy Oncotarget 2017 8 34 57813 10.18632/oncotarget.18175 28915713
Fu, Y. et al. The reverse Warburg effect is likely to be an Achilles’ heel of cancer that can be exploited for cancer therapy. Oncotarget 8(34), 57813 (2017).28915713 10.18632/oncotarget.18175
12. Galluzzi L Kepp O Kroemer G Reverse Warburg: Straight to cancer Cell Cycle 2012 11 6 1059 1059 10.4161/cc.11.6.19746 22343921
Galluzzi, L., Kepp, O. & Kroemer, G. Reverse Warburg: Straight to cancer. Cell Cycle 11(6), 1059–1059 (2012).22343921 10.4161/cc.11.6.19746
13. Ždralević, M. et al. Disrupting the ‘Warburg effect’re-routes cancer cells to OXPHOS offering a vulnerability point via ‘ferroptosis’-induced cell death. In Advances in Biological Regulation (2017).
14. Beloueche-Babari, M. et al. MCT1 inhibitor AZD3965 increases mitochondrial metabolism, facilitating combination therapy and non-invasive magnetic resonance spectroscopy. Cancer Res. canres. 2686.2016 (2017).
15. Hong CS MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4 Cell Rep. 2016 14 7 1590 1601 10.1016/j.celrep.2016.01.057 26876179
Hong, C. S. et al. MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4. Cell Rep. 14(7), 1590–1601 (2016).26876179 10.1016/j.celrep.2016.01.057
16. Brown JM Tumor hypoxia in cancer therapy Methods Enzymol. 2007 435 295 321 10.1016/S0076-6879(07)35015-5
Brown, J. M. Tumor hypoxia in cancer therapy. Methods Enzymol. 435, 295–321 (2007).10.1016/S0076-6879(07)35015-5
17. Lara PC Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression Radiat. Oncol. 2009 4 1 29 10.1186/1748-717X-4-29 19660100
Lara, P. C. et al. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression. Radiat. Oncol. 4(1), 29 (2009).19660100 10.1186/1748-717X-4-29
18. Rockwell S Hypoxia and radiation therapy: Past history, ongoing research, and future promise Curr. Mol. Med. 2009 9 4 442 458 10.2174/156652409788167087 19519402
Rockwell, S. et al. Hypoxia and radiation therapy: Past history, ongoing research, and future promise. Curr. Mol. Med. 9(4), 442–458 (2009).19519402 10.2174/156652409788167087
19. Walenta S High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers Cancer Res. 2000 60 4 916 921 10706105
Walenta, S. et al. High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer Res. 60(4), 916–921 (2000).10706105
20. Xu HN Is higher lactate an indicator of tumor metastatic risk? A pilot MRS study using hyperpolarized 13C-pyruvate Acad. Radiol. 2014 21 2 223 231 10.1016/j.acra.2013.11.014 24439336
Xu, H. N. et al. Is higher lactate an indicator of tumor metastatic risk? A pilot MRS study using hyperpolarized 13C-pyruvate. Acad. Radiol. 21(2), 223–231 (2014).24439336 10.1016/j.acra.2013.11.014
21. Goetze K Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release Int. J. Oncol. 2011 39 2 453 463 21617859
Goetze, K. et al. Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release. Int. J. Oncol. 39(2), 453–463 (2011).21617859
22. Romero-Garcia S Lactate contribution to the tumor microenvironment: Mechanisms, effects on immune cells and therapeutic relevance Front. Immunol. 2016 7 52 10.3389/fimmu.2016.00052 26909082
Romero-Garcia, S. et al. Lactate contribution to the tumor microenvironment: Mechanisms, effects on immune cells and therapeutic relevance. Front. Immunol. 7, 52 (2016).26909082 10.3389/fimmu.2016.00052
23. de la Cruz-López KG Lactate in the regulation of tumor microenvironment and therapeutic approaches Front. Oncol. 2019 9 1143 10.3389/fonc.2019.01143 31737570
de la Cruz-López, K. G. et al. Lactate in the regulation of tumor microenvironment and therapeutic approaches. Front. Oncol. 9, 1143 (2019).31737570 10.3389/fonc.2019.01143
24. Kefayat, A. et al. Alive attenuated Salmonella as a cargo shuttle for smart carrying of gold nanoparticles to tumor hypoxic regions. J. Drug Target. 1–28 (2018).
25. Kaimala S Attenuated Bacteria as immunotherapeutic tools for cancer treatment Frontiers in oncology 2018 8 136 10.3389/fonc.2018.00136 29765907
Kaimala, S. et al. Attenuated Bacteria as immunotherapeutic tools for cancer treatment. Frontiers in oncology 8, 136 (2018).29765907 10.3389/fonc.2018.00136
26. Li B Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo Acta Pharm. Sin. B 2012 2 6 562 568 10.1016/j.apsb.2012.10.006
Li, B. et al. Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo. Acta Pharm. Sin. B 2(6), 562–568 (2012).10.1016/j.apsb.2012.10.006
27. Song S Vuai MS Zhong M The role of bacteria in cancer therapy–enemies in the past, but allies at present Infect. Agents Cancer 2018 13 1 9 10.1186/s13027-018-0180-y
Song, S., Vuai, M. S. & Zhong, M. The role of bacteria in cancer therapy–enemies in the past, but allies at present. Infect. Agents Cancer 13(1), 9 (2018).10.1186/s13027-018-0180-y
28. Leschner S Weiss S Salmonella—Allies in the fight against cancer J. Mol. Med. 2010 88 8 763 773 10.1007/s00109-010-0636-z 20526574
Leschner, S. & Weiss, S. Salmonella—Allies in the fight against cancer. J. Mol. Med. 88(8), 763–773 (2010).20526574 10.1007/s00109-010-0636-z
29. Danino T Measuring growth and gene expression dynamics of tumor-targeted S. typhimurium bacteria J. Vis. Exp. JoVE 2013 77 e50540
Danino, T. et al. Measuring growth and gene expression dynamics of tumor-targeted S. typhimurium bacteria. J. Vis. Exp. JoVE 77, e50540 (2013).
30. Yu B Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella typhimurium strain Sci. Rep. 2012 2 436 10.1038/srep00436 22666539
Yu, B. et al. Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella typhimurium strain. Sci. Rep. 2, 436 (2012).22666539 10.1038/srep00436
31. Van Mellaert L Barbé S Anné J Clostridium spores as anti-tumour agents TRENDS Microbiol. 2006 14 4 190 196 10.1016/j.tim.2006.02.002 16500103
Van Mellaert, L., Barbé, S. & Anné, J. Clostridium spores as anti-tumour agents. TRENDS Microbiol. 14(4), 190–196 (2006).16500103 10.1016/j.tim.2006.02.002
32. Vernon LF William Bradley Coley, MD, and the phenomenon of spontaneous regression ImmunoTargets Therapy 2018 7 29 10.2147/ITT.S163924 29719818
Vernon, L. F. William Bradley Coley, MD, and the phenomenon of spontaneous regression. ImmunoTargets Therapy 7, 29 (2018).29719818 10.2147/ITT.S163924
33. Zheng JH Min J-J Targeted cancer therapy using engineered Salmonella typhimurium Chonnam Med. J. 2016 52 3 173 184 10.4068/cmj.2016.52.3.173 27689027
Zheng, J. H. & Min, J.-J. Targeted cancer therapy using engineered Salmonella typhimurium. Chonnam Med. J. 52(3), 173–184 (2016).27689027 10.4068/cmj.2016.52.3.173
34. McCarthy EF The toxins of William B. Coley and the treatment of bone and soft-tissue sarcomas Iowa Orthop. J. 2006 26 154 16789469
McCarthy, E. F. The toxins of William B. Coley and the treatment of bone and soft-tissue sarcomas. Iowa Orthop. J. 26, 154 (2006).16789469
35. Zheng H-C The molecular mechanisms of chemoresistance in cancers Oncotarget 2017 8 35 59950 10.18632/oncotarget.19048 28938696
Zheng, H.-C. The molecular mechanisms of chemoresistance in cancers. Oncotarget 8(35), 59950 (2017).28938696 10.18632/oncotarget.19048
36. Willers H Basic mechanisms of therapeutic resistance to radiation and chemotherapy in lung cancer Cancer J. 2013 19 3 200 10.1097/PPO.0b013e318292e4e3 23708066
Willers, H. et al. Basic mechanisms of therapeutic resistance to radiation and chemotherapy in lung cancer. Cancer J. 19(3), 200 (2013).23708066 10.1097/PPO.0b013e318292e4e3
37. Nallar SC Xu D-Q Kalvakolanu DV Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges Cytokine 2017 89 160 172 10.1016/j.cyto.2016.01.002 26778055
Nallar, S. C., Xu, D.-Q. & Kalvakolanu, D. V. Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges. Cytokine 89, 160–172 (2017).26778055 10.1016/j.cyto.2016.01.002
38. Liu S Tumor-targeting bacterial therapy: A potential treatment for oral cancer Oncol. Lett. 2014 8 6 2359 2366 10.3892/ol.2014.2525 25364397
Liu, S. et al. Tumor-targeting bacterial therapy: A potential treatment for oral cancer. Oncol. Lett. 8(6), 2359–2366 (2014).25364397 10.3892/ol.2014.2525
39. Luo C-H Bacteria-mediated hypoxia-specific delivery of nanoparticles for tumors imaging and therapy Nano Lett. 2016 16 6 3493 3499 10.1021/acs.nanolett.6b00262 27148804
Luo, C.-H. et al. Bacteria-mediated hypoxia-specific delivery of nanoparticles for tumors imaging and therapy. Nano Lett. 16(6), 3493–3499 (2016).27148804 10.1021/acs.nanolett.6b00262
40. Jiang S-N Inhibition of tumor growth and metastasis by a combination of Escherichia coli–mediated cytolytic therapy and radiotherapy Mol. Therapy 2010 18 3 635 642 10.1038/mt.2009.295
Jiang, S.-N. et al. Inhibition of tumor growth and metastasis by a combination of Escherichia coli–mediated cytolytic therapy and radiotherapy. Mol. Therapy 18(3), 635–642 (2010).10.1038/mt.2009.295
41. Roberts NJ Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses Sci. Transl. Med. 2014 6 249 249ra111-249ra111 10.1126/scitranslmed.3008982
Roberts, N. J. et al. Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Sci. Transl. Med. 6(249), 249ra111-249ra111 (2014).10.1126/scitranslmed.3008982
42. Cronin M Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors Mol. Therapy 2010 18 7 1397 1407 10.1038/mt.2010.59
Cronin, M. et al. Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors. Mol. Therapy 18(7), 1397–1407 (2010).10.1038/mt.2010.59
43. Yazawa K Bifidobacterium longum as a delivery system for cancer gene therapy: Selective localization and growth in hypoxic tumors Cancer Gene Therapy 2000 7 2 269 10.1038/sj.cgt.7700122 10770636
Yazawa, K. et al. Bifidobacterium longum as a delivery system for cancer gene therapy: Selective localization and growth in hypoxic tumors. Cancer Gene Therapy 7(2), 269 (2000).10770636 10.1038/sj.cgt.7700122
44. Rosenberg SA Spiess PJ Kleiner DE Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium J. Immunother. 2002 25 3 218 10.1097/00002371-200205000-00004 12000863
Rosenberg, S. A., Spiess, P. J. & Kleiner, D. E. Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. J. Immunother. 25(3), 218 (2002).12000863 10.1097/00002371-200205000-00004
45. Wood LM Paterson Y Attenuated Listeria monocytogenes: A powerful and versatile vector for the future of tumor immunotherapy Front. Cell. Infect. Microbiol. 2014 4 51 10.3389/fcimb.2014.00051 24860789
Wood, L. M. & Paterson, Y. Attenuated Listeria monocytogenes: A powerful and versatile vector for the future of tumor immunotherapy. Front. Cell. Infect. Microbiol. 4, 51 (2014).24860789 10.3389/fcimb.2014.00051
46. Walsh JC The clinical importance of assessing tumor hypoxia: Relationship of tumor hypoxia to prognosis and therapeutic opportunities Antioxid. Redox Signal. 2014 21 10 1516 1554 10.1089/ars.2013.5378 24512032
Walsh, J. C. et al. The clinical importance of assessing tumor hypoxia: Relationship of tumor hypoxia to prognosis and therapeutic opportunities. Antioxid. Redox Signal. 21(10), 1516–1554 (2014).24512032 10.1089/ars.2013.5378
47. Torres W Bacteria in cancer therapy: Beyond immunostimulation J. Cancer Metastasis Treat 2018 4 4 10.20517/2394-4722.2017.49
Torres, W. et al. Bacteria in cancer therapy: Beyond immunostimulation. J. Cancer Metastasis Treat 4, 4 (2018).10.20517/2394-4722.2017.49
48. Stern C Induction of CD 4+ and CD 8+ anti-tumor effector T cell responses by bacteria mediated tumor therapy Int. J. Cancer 2015 137 8 2019 2028 10.1002/ijc.29567 25868911
Stern, C. et al. Induction of CD 4+ and CD 8+ anti-tumor effector T cell responses by bacteria mediated tumor therapy. Int. J. Cancer 137(8), 2019–2028 (2015).25868911 10.1002/ijc.29567
49. Camacho EM Engineering Salmonella as intracellular factory for effective killing of tumour cells Sci. Rep. 2016 6 30591 10.1038/srep30591 27464652
Camacho, E. M. et al. Engineering Salmonella as intracellular factory for effective killing of tumour cells. Sci. Rep. 6, 30591 (2016).27464652 10.1038/srep30591
50. Mashima I Identification of Veillonella species in the tongue biofilm by using a novel one-step polymerase chain reaction method PloS One 2016 11 6 e0157516 10.1371/journal.pone.0157516 27326455
Mashima, I. et al. Identification of Veillonella species in the tongue biofilm by using a novel one-step polymerase chain reaction method. PloS One 11(6), e0157516 (2016).27326455 10.1371/journal.pone.0157516
51. Ng SK Hamilton IR Lactate metabolism by Veillonella parvula J. Bacteriol. 1971 105 3 999 1005 10.1128/jb.105.3.999-1005.1971 4323300
Ng, S. K. & Hamilton, I. R. Lactate metabolism by Veillonella parvula. J. Bacteriol. 105(3), 999–1005 (1971).4323300 10.1128/jb.105.3.999-1005.1971
52. Bindels LB Gut microbiota-derived propionate reduces cancer cell proliferation in the liver Br. J. Cancer 2012 107 8 1337 10.1038/bjc.2012.409 22976799
Bindels, L. B. et al. Gut microbiota-derived propionate reduces cancer cell proliferation in the liver. Br. J. Cancer 107(8), 1337 (2012).22976799 10.1038/bjc.2012.409
53. Chang X Propionate-producing Veillonella parvula regulates the malignant properties of tumor cells of OSCC Med. Oncol. 2023 40 3 98 10.1007/s12032-023-01962-6 36808012
Chang, X. et al. Propionate-producing Veillonella parvula regulates the malignant properties of tumor cells of OSCC. Med. Oncol. 40(3), 98 (2023).36808012 10.1007/s12032-023-01962-6
54. Kefayat, A., Ghahremani, F. & Rostami, S. Veillonella parvula: A strictly anaerobic bacterium with high efficacy for safe and specific tumor targeting and colonization. bioRxiv 2021.05.10.443531 (2021).
55. Varshosaz J Folated synperonic-cholesteryl hemisuccinate polymeric micelles for the targeted delivery of docetaxel in melanoma BioMed Res. Int. 2015 2015 746093 10.1155/2015/746093 25839040
Varshosaz, J. et al. Folated synperonic-cholesteryl hemisuccinate polymeric micelles for the targeted delivery of docetaxel in melanoma. BioMed Res. Int. 2015, 746093 (2015).25839040 10.1155/2015/746093
56. Ibrahim K Histopathology of the liver, kidney, and spleen of mice exposed to gold nanoparticles Molecules 2018 23 8 1848 10.3390/molecules23081848 30044410
Ibrahim, K. et al. Histopathology of the liver, kidney, and spleen of mice exposed to gold nanoparticles. Molecules 23(8), 1848 (2018).30044410 10.3390/molecules23081848
57. Engbaek K Johansen KS Jensen ME A new technique for Gram staining paraffin-embedded tissue J. Clin. Pathol. 1979 32 2 187 190 10.1136/jcp.32.2.187 86548
Engbaek, K., Johansen, K. S. & Jensen, M. E. A new technique for Gram staining paraffin-embedded tissue. J. Clin. Pathol. 32(2), 187–190 (1979).86548 10.1136/jcp.32.2.187
58. Brown RC Hopps HC Staining of bacteria in tissue sections: A reliable Gram stain method Am. J. Clin. Pathol. 1973 60 2 234 240 10.1093/ajcp/60.2.234 4124318
Brown, R. C. & Hopps, H. C. Staining of bacteria in tissue sections: A reliable Gram stain method. Am. J. Clin. Pathol. 60(2), 234–240 (1973).4124318 10.1093/ajcp/60.2.234
59. Kefayat A c-phycocyanin: A natural product with radiosensitizing property for enhancement of colon cancer radiation therapy efficacy through inhibition of COX-2 expression Sci. Rep. 2019 9 1 19161 10.1038/s41598-019-55605-w 31844085
Kefayat, A. et al. c-phycocyanin: A natural product with radiosensitizing property for enhancement of colon cancer radiation therapy efficacy through inhibition of COX-2 expression. Sci. Rep. 9(1), 19161 (2019).31844085 10.1038/s41598-019-55605-w
60. Felgner S Tumour-targeting bacteria-based cancer therapies for increased specificity and improved outcome Microb. Biotechnol. 2017 10 5 1074 1078 10.1111/1751-7915.12787 28771926
Felgner, S. et al. Tumour-targeting bacteria-based cancer therapies for increased specificity and improved outcome. Microb. Biotechnol. 10(5), 1074–1078 (2017).28771926 10.1111/1751-7915.12787
61. Kramer MG Bacterial therapy of cancer: Promises, limitations, and insights for future directions Front. Microbiol. 2018 9 16 10.3389/fmicb.2018.00016 29472896
Kramer, M. G. et al. Bacterial therapy of cancer: Promises, limitations, and insights for future directions. Front. Microbiol. 9, 16 (2018).29472896 10.3389/fmicb.2018.00016
62. Kefayat A Ultra-small but ultra-effective: Folic acid-targeted gold nanoclusters for enhancement of intracranial glioma tumors’ radiation therapy efficacy Nanomedicine 2019 16 173 184 10.1016/j.nano.2018.12.007 30594659
Kefayat, A. et al. Ultra-small but ultra-effective: Folic acid-targeted gold nanoclusters for enhancement of intracranial glioma tumors’ radiation therapy efficacy. Nanomedicine 16, 173–184 (2019).30594659 10.1016/j.nano.2018.12.007
63. Kefayat A Investigation of different targeting decorations effect on the radiosensitizing efficacy of albumin-stabilized gold nanoparticles for breast cancer radiation therapy Eur. J. Pharm. Sci. 2019 130 225 233 10.1016/j.ejps.2019.01.037 30711685
Kefayat, A. et al. Investigation of different targeting decorations effect on the radiosensitizing efficacy of albumin-stabilized gold nanoparticles for breast cancer radiation therapy. Eur. J. Pharm. Sci. 130, 225–233 (2019).30711685 10.1016/j.ejps.2019.01.037
64. Sun X Kaufman PD Ki-67: More than a proliferation marker Chromosoma 2018 127 175 186 10.1007/s00412-018-0659-8 29322240
Sun, X. & Kaufman, P. D. Ki-67: More than a proliferation marker. Chromosoma 127, 175–186 (2018).29322240 10.1007/s00412-018-0659-8
65. Walenta, S. & Mueller-Klieser, W. F. Lactate: Mirror and Motor of Tumor Malignancy. In Seminars in Radiation Oncology (Elsevier, 2004).
66. Bonuccelli G Ketones and lactate “fuel” tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism Cell Cycle 2010 9 17 3506 3514 10.4161/cc.9.17.12731 20818174
Bonuccelli, G. et al. Ketones and lactate “fuel” tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism. Cell Cycle 9(17), 3506–3514 (2010).20818174 10.4161/cc.9.17.12731
67. Serganova, I. et al. Metabolic imaging: A link between lactate dehydrogenase A, lactate and tumor phenotype. Clin. Cancer Res. clincanres.0397.2011 (2011).
68. Mowday A Advancing clostridia to clinical trial: Past lessons and recent progress Cancers 2016 8 7 63 10.3390/cancers8070063 27367731
Mowday, A. et al. Advancing clostridia to clinical trial: Past lessons and recent progress. Cancers 8(7), 63 (2016).27367731 10.3390/cancers8070063
69. Strach M Sepsis caused by Veillonella parvula infection in a 17-year-old patient with X-linked agammaglobulinemia (Bruton’s disease) J. Clin. Microbiol. 2006 44 7 2655 2656 10.1128/JCM.00467-06 16825407
Strach, M. et al. Sepsis caused by Veillonella parvula infection in a 17-year-old patient with X-linked agammaglobulinemia (Bruton’s disease). J. Clin. Microbiol. 44(7), 2655–2656 (2006).16825407 10.1128/JCM.00467-06
70. Li J A new treatment of sepsis caused by Veillonella parvula: A case report and literature review J. Clin. Pharm. Ther. 2017 42 5 649 652 10.1111/jcpt.12559 28543519
Li, J. et al. A new treatment of sepsis caused by Veillonella parvula: A case report and literature review. J. Clin. Pharm. Ther. 42(5), 649–652 (2017).28543519 10.1111/jcpt.12559
71. Doherty JR Cleveland JL Targeting lactate metabolism for cancer therapeutics J. Clin. Investig. 2013 123 9 3685 3692 10.1172/JCI69741 23999443
Doherty, J. R. & Cleveland, J. L. Targeting lactate metabolism for cancer therapeutics. J. Clin. Investig. 123(9), 3685–3692 (2013).23999443 10.1172/JCI69741
72. Nejman D The human tumor microbiome is composed of tumor type–specific intracellular bacteria Science 2020 368 6494 973 980 10.1126/science.aay9189 32467386
Nejman, D. et al. The human tumor microbiome is composed of tumor type–specific intracellular bacteria. Science 368(6494), 973–980 (2020).32467386 10.1126/science.aay9189
