Holistic Cancer Treatment: Low Dose Chemo – Part 5

Holistic Cancer Treatment: Low Dose Chemo – Part 5

Low Dose Chemo Part 5 – Holistic Cancer Treatment – An Oasis of Healing

This blog post is part two of a two part series on low-dose, metronomic chemotherapy and angiogenesis. I encourage you to read part one here.

I read some poetry, but I am no poet, that is for sure. A very famous line from one of Elizabeth Barrett Browning’s more famous poems is “Let me count the ways.” This line was a reference to love. What does the Elizabeth Barrett Browning quote have to do with low-dose, metronomic chemotherapy, because love sure has no connection? They quite obviously have nothing to do with each other. It will, however, help me count and discuss the many ways that low-dose, metronomic chemotherapy can inhibit angiogenesis.

Let’s not waste anytime and jump right in.

How does low-dose, metronomic chemotherapy inhibit tumor angiogenesis?

• Low-dose chemotherapy Reduces Vascular Epithelial Growth Factor (VEGF) signaling ^{[1] [2] [3]}.

What is VEGF? Vascular Endothelial Growth Factor is a key signal in the stimulation of angiogenesis. The name gives it’s function away as VEGF stimulates endothelial cells’ growth to support new vascular growth. But what exactly is the role of VEGF? Is it all negative? First, VEGF doesn’t play only a negative role, i.e., stimulation of angiogenesis in cancer. Vascular endothelial growth factor is a normal part of a developing pregnancy. Think of all the vascularization and new blood vessel growth required in the placenta alone! Vascular endothelial growth factor also plays an important role in the neovascularization, angiogenesis process required for healing on a day to day process. Can anyone say paper cuts? But as with so many things in the body, cancer hijacks normal, good day to day pathway signaling required for health and wellness to support its ill-intentioned survival and growth—that is bad.

Increased VEGF signaling in cancer plays a central role in stimulating angiogenesis in support of cancer growth. In cancer, the stimulation of VEGF occurs in a multitude of ways:

• HIF-1alpha stimulates VEGF ^{[4] [5]}
• NF-κB inflammatory genetic transcription factor stimulates VEGF ^[6]
• Inflammatory cytokines (IL-1, IL-6, IL-8, IL-10, TNF-alpha, TGF-beta) stimulates VEGF ^{[7] [8]}
• Cox-2 expression stimulates VEGF ^{[9] [10]}
• Growth factors (Insulin Growth Factor-1, Epidermal Growth Factor, Platelet-derived Growth Factor, Fibroblast Growth Factor) stimulates VEGF ^{[11] [12]}
• Oncogene (BCR-ABL, Ras) activation stimulates VEGF ^[13]
• Tumor Suppressor gene (p53) inhibition stimulates VEGF

I could go on and on. More than the fact that these signals can stimulate angiogenesis, they stimulate defective angiogenesis ^[14]. Also, remember that maximum to tolerated toxicity chemotherapy dosing produces cytokine storm, which promotes metastasis; and one of the critical mechanisms is through angiogenesis. This point alone highlights the stark contrast between low-dose, metronomic chemotherapy, and maximum to tolerated toxicity chemotherapy. Cancer is a multifactorial process and highlights the failing paradigm of maximum to tolerated toxicity dosing approach.

• Low-dose, metronomic chemotherapy inhibits HIF-1alpha.

 Some things just go together: where there is smoke, there is fire, song and dance, pencil and paper, salt and pepper. So too is the relationship between cancer and hypoxia. Hypoxia is the low oxygen environment that is so often found in the cancer tumor microenvironment. In many ways, hypoxia opens the door for angiogenesis through the signal of Hypoxia-Inducible Factor-1alpha (HIF-1alpha). Hypoxia Inducible Factor-1alpha is a critical to the process of angiogenesis. More than angiogenesis, HIF-1alpha expression is an essential step towards tumor growth and metastasis. Other than tumor growth and metastasis, which is very important, what is the relevance to this current discussion? The answer is quite direct. According to published science, low-dose, metronomic chemotherapy inhibits HIF-1alpha and thus angiogenesis ^{[15] [16]}.

• Low-dose, metronomic chemotherapy inhibits Hypoxia-Inducible Factor-1 alpha (HIF-1alpha) VEGF signaling.

Hypoxia, which is prevalent in the tumor microenvironment of cancer, drives HIF-1alpha signaling. Hypoxia-Inducible Factor-1alpha signaling plays a central role in this stimulation of angiogenesis in the cancer process ^{[17] [18]}. If you think about it, this connection makes perfect sense—a growing tumor with hypoxia in the local tumor microenvironment would stimulate HIF-1alpha to support an increase in new vessel growth to counter the hypoxia and meet the increasing metabolic demand of growth. However, as previously stated, the new vascular growth in cancer is very defective. Low-dose, metronomic chemotherapy inhibits the HIF-1alpha—> VEGF—> angiogenesis—> tumor growth—> metastasis cascade ^{2 [19] [20]}.

• Low-dose chemotherapy inhibits T regulator cells ^{[21] [22]}.

 The weeds only get thicker, so hang with me. What the heck are T regulator cells? To be specific, T regulator cells are a type of T lymphocyte that regulate and suppress effector (or attack) T cells and antigen-presenting cells. Lymphocytes are critical immune cells used by the body against cancer cells. Think of the effector T cells as doing the day to day combat against invaders, both foreign (bacteria, virus’…) and domestic (cancer). CD8+ T cells, called cytotoxic T lymphocytes, are one such example. Then there are antigen-presenting cells. Antigen-presenting cells are like the on-field commanders identifying the enemy and helping direct the army, like T effector cells, to seek and destroy the enemy—cancer. T regulator cells suppress both the coordination and hand-to-hand combat effect of the immune system against invaders, both foreign and domestic. Thus, T regulator cells are immunosuppressive. If the body’s defenses are misdirected, i.e., autoimmune disease, this is a good control mechanism to prevent self-destruction. However, like many pathways, cancer high-jacks this regulation signaling pathway to suppress the immune system defenses leaving cancer without opposition, free to grow and spread. Research has shown that an increase in T regulator cells is associated with increased treatment resistance and tumor progression ^[23]. In the context of cancer, this is not good.

But, what is the relevance to low-dose, metronomic chemotherapy, and angiogenesis? Well, I am so glad you asked! T regulator cell activity plays a crucial role in angiogenesis. Remember, angiogenesis is a normal part of the healing process, but cancer high-jacks it. Evidence exists to show that low-dose, metronomic chemotherapy inhibits and depletes T regulator lymphocytes, which inhibits angiogenesis and cancer’s lifeblood for survival ^{[24] [25] [26] [27]}. But there is more. Low-dose metronomic chemotherapy increases the presence of cancer cell destroying, cytotoxic, CD8+ T lymphocytes, and NK cells in the local tumor environment ^{24 [28]}. That is a double anticancer whammy! Thank you low-dose, metronomic chemotherapy.

• Maximum tolerated chemotherapy increases T regulator cells in TME?

As described above, T regulator cells are a subset of T lymphocytes that actively suppress the immune system response ^{[29] [30]}. The T regulator suppression of the immune system is a crucial step in cancer’s ability to escape the immune system through immune tolerance, called immune escape, which leads to the metastatic spread of cancer ^[31].

In steps the paradigm and regimen of maximum to tolerated toxicity chemotherapy in the treatment of cancer. In the previous bullet point above, I pointed out how low-dose, metronomic chemotherapy can suppress T regulator cells, which removes the cloak of immune suppression to free the immune system to do its job—kill cancer cells. Instead of following the science here, most conventional medicine treads water in the shallow end of the pool with statements of “The mechanistic understanding of why low dose metronomic chemotherapy regimens are generally more immune-stimulating is not entirely clear” ³², or the often repeated and never achieved—more studies are needed. But, when one looks at the evidence pool’s deeper end, maximum to tolerated toxicity chemotherapy (MTTC) increases immune-suppressing with T regulator cells. But there is more; maximum to tolerated toxicity chemotherapy also depresses effector CD8+ cells, suppresses CD4+ T cells, suppresses Natural Killer (NK) cells, and increases cancer-associated fibroblasts ^{[32] [33] [34]}. This point is important because an increase in T regulator cells throughout the body and the tumor microenvironment is associated with tumor progression, worse prognosis, and worse outcomes ^{27 [35]}.

This MTTC induced immune tolerance occurs throughout the body, but the bulls-eye of this activity is within the tumor microenvironment. In contrast, as discussed in the point above, low dose, metronomic chemotherapy suppresses the immune-suppressing T regulator cells; but increases anticancer effector T cells and activates natural killer cells at the same time. All these immune effects are throughout but are laser-focused within the tumor microenvironment. What once was considered the answer to cancer is really just the answer to how maximum to tolerated toxicity chemotherapy can cause cancer to spread. Amazing!

• Low-dose chemotherapy inhibits TGF-beta.

The next logical step after T regulator lymphocytes is Transforming Growth Factor-beta (TGF-beta). T regulator lymphocytes secrete TGF-beta. Transforming Growth Factor-beta is an immune system inflammatory cytokine signal responsible for regulating cell growth and differentiation. This description is more eloquently stated in a 2012 BMC Immunology journal article,

“Transforming growth factor-beta (TGFβ) is a multi-functional cytokine that regulates cell proliferation, differentiation, migration, and survival. It plays a critical role in development, wound healing, and immune responses through its regulatory effects on many cell types…” ^[36]

 It is easy to see why this would be so important in cancer due to its unregulated, pro-growth, and poorly de-differentiated nature. In contrast, healthy, normal cells are regulated in growth and differentiated in development. In the presence of cancer, this inflammatory cytokine signal helps to form a circle of immune system destruction that protects cancer from the same immune system designed for its destruction. Yes, you did read that right. Cancer co-opts and uses the immune system to protect itself from the immune system. Again, the majority of this activity occurs in the tumor microenvironment. Transforming Growth Factor-beta is the master on switch for T regulator cell development and activation to initiate this cancer protection from the immune system. The activated T-regulator cells, in turn, secrete TGF-beta to suppress the effector T-cells killing of cancer cells ^{[37] [38]}. The result is a circle of immune suppression that leads to cancer growth using the tools already at its disposal.

What is the connection between low-dose metronomic chemotherapy, TGF-beta, and angiogenesis? Hypoxia in the tumor microenvironment is the process that drives tumor angiogenesis. Stroke research has shown that hypoxia post-stroke causes angiogenesis via an increase in TGF-beta signaling ^[39]. In addition, post-transplant surgery angiogenesis is activated by hypoxia TGF-beta signaling ^[40]. In highly vascular cancer types, glioblastoma and hepatocellular, TGF-beta signaling has been shown to stimulate angiogenesis ^[41]. Some cancer types are more vascular than others. I am oversimplifying a very complicated process. But taking this to its logical conclusion, inhibition of TGF-beta signaling in cancer inhibits angiogenesis ^{[42] [43] [44]}. As mentioned previously, low-dose metronomic chemotherapy inhibits T regulator cell activity in the tumor microenvironment, decreases VEGF, and increases anti-cancer cytotoxic CD8+ T lymphocytes in the tumor microenvironment. The only conclusion possible is that TGF-beta signaling is reduced by low-dose, metronomic chemotherapy. However, there are no such studies that I have been able to find that specifically support the previous sentence. However, the logical conclusion is that metronomic chemotherapy, through its decrease in T regulator cells, does, in fact, inhibit TGF-beta signaling because T regulator cells secrete TGF-beta. The counter is also accurate—an increase in CD8+ T cell anti-cancer activity in the tumor microenvironment is also evidence of a decrease in TGF-beta because an increase in CD8+ T cells equates to a reduction in T regulator cells which secrete TGF-beta. Hopefully, a future study will look at this. Maybe this article can be the impetus for that study; or if you know of a study that I have missed, please send my way.

• Low dose chemotherapy inhibits M2 Macrophage polarization ^[45].

T regulator cells stimulate angiogenesis, T regulator cells produce TGF-beta, and TGF-beta signaling produces angiogenesis. The next step in signaling is Transforming Growth Factor-beta, which stimulates M2 macrophage polarization in the tumor microenvironment.

I have previously talked about Tumor Associated Macrophages (TAMs), M1 macrophage, and M2 macrophage polarization (think movement) in the tumor microenvironment, so I don’t want to unpack all that again here. But, to hit the high notes, M2 macrophage polarization in the tumor microenvironment is pro cancer growth and spread; in contrast, M1 macrophage polarization is anti-cancer. That being said, TGF-beta signaling in the tumor microenvironment has been shown to promote M2 macrophage polarization, and M2 macrophages have been shown to increase TGF-beta signaling ^[46].

It is essential to understand the significance of M2 macrophage polarization in the tumor microenvironment. Maximum tolerated to toxicity chemotherapy dosing has been shown to promote cancer cell physical escape and immune escape. It does this in a variety of ways. First, the cytokine storm (increase in IL-6) that is induced by maximum to tolerated toxicity chemotherapy dosing recruits macrophages, called Tumor Associated Macrophages (TAMs), into the local tumor microenvironment. In the hypoxic conditions encountered in the local tumor microenvironment, M2 macrophage activation, M1 to M2 macrophage polarization, and M2 macrophage stabilization dominates. Transforming Growth Factor-beta is intimately involved in this TAM recruitment and resultant M2 macrophage polarization ^{[47] [48]}. In a comparable way to pregnancy, cancer stimulates immune suppression to provide immuno-tolerance to escape immune destruction. The two ‘escapes’ (physical and immune) are critical steps in the mobilization and movement of cancer cells from the primary tumor to distant sites in the metastatic cancer process—all of which is driven, in part, by M2 macrophages from maximum to tolerated toxicity dosing of chemotherapy ^{[49] [50] [51]}.

Transforming Growth Factor-beta is one of the mechanisms by which M2 polarization in the tumor microenvironment occurs. Research has shown that activation of TAMs into an M2 macrophage dominance occurs, in part, from TGF-beta signaling. I also failed to mention that M2 macrophages also secrete TGF-beta ^[52]. As so often is found in cancer, self-destruction begets more self-destruction. It is this very signaling that promotes cancer proliferation, physical escape, and then immune escape.

What is the relevance to low-dose, metronomic chemotherapy, and angiogenesis? First, the current science suggests low-dose, metronomic chemotherapy does not stimulate the cytokine storm induced metastasis as does maximum to tolerated toxicity chemotherapy ^{33 [53]}. Second, it is the low-dose, metronomic approach to the dosing of chemotherapy that inhibits the cascade of T regulator cell activation—>TGF-beta production—>M1 to M2 polarization—>cancer cell physical escape—>cancer cell immune escape—>metastasis. The third point, maximum to tolerated toxicity chemotherapy, induces rapid resistance to treatment ^[54]. Last, though different but very similar to M1 to M2 polarization, low-dose, metronomic chemotherapy has been shown to prevent regular dendritic cell’s polarization into immunosuppressive regulatory dendritic cells ^{[55] [56] [57]}. Why was any of this biochemistry baptism necessary or important? Ninety percent of cancer morbidity and mortality occurs as a result of cancer metastasis. Any treatment that causes the metastatic spread of cancer is not an effective or acceptable therapy.

This blog post was quite the journey through the biochemical weeds. Thanks for sticking with me through this post. This theme could go on and on. For example, low-dose, metronomic chemotherapy inhibits the pro-angiogenic mobilization of Endothelial Progenitor Cells (EPC) ⁵³ from bone morrow essential to vasculogenesis and stimulates the production of the anti-angiogenic Thrombospondin-1 ^{[58] [59] [60]}, both of which inhibit carcinogenic angiogenesis. I know it seems like I am piling on, but that is the evidence.

Is there evidence that the Holistic, Integrative cancer treatment with low-dose, metronomic chemotherapy inhibits cancer angiogenesis? I hope I have been able to put that question to rest with the evidence in this post.

In conclusion, let’s boil this down to the basic take-home conclusions. The take-home is that low-dose, metronomic chemotherapy inhibits cancer angiogenesis. And it does this in a wide variety of ways. The biochemistry doesn’t lie. In contrast, maximum tolerated to toxicity chemotherapy can stimulate cytokine storm, angiogenesis, and metastasis. Now that is a stark contrast!

I started this two-part blogpost series on low-dose, metronomic chemotherapy and angiogenesis with a quote from Thomas Kuhn, so let me wrap this up with another quote:

“Rather than being an interpreter, the scientist who embraces a new paradigm is like the man wearing inverting lenses”

“Literally as well as metaphorically, the man accustomed to inverting lenses has undergone a revolutionary transformation of vision.”

—Thomas Kuhn

In the paradigm of cancer treatment, it is time for a revolutionary transformation of thought, vision, and action for the benefit of individuals and families effected by cancer.

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^[1] Lau DH, Xue L, Young LJ, Burke PA, Cheung AT. Paclitaxel (Taxol): an inhibitor of angiogenesis in a highly vascularized transgenic breast cancer. Cancer Biother Radiopharm. 1999;14: 31-36.

^[2] Zeng J, Yang L, Huang F, Hong T, He Z, Lei J, Sun H, Lu Y, Hao X. The metronomic therapy with prednisone, etoposide, and cyclophosphamide reduces the serum levels of VEGF and circulating endothelial cells and improves response rates and progression-free survival in patients with relapsed or refractory non-Hodgkin’s lymphoma. Cancer Chemother Pharmacol. 2016;78:801-808.

^[3] Simsek C, Esin E, Yalcin S. Metronomic Chemotherapy: A Systematic Review of the Literature and Clinical Experience. J Oncol. 2019;2019:5483791. Published 2019 Mar 20. doi:10.1155/2019/5483791

^[4] Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676.

^[5] Gordan JD, Simon MC. Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev. 2007;17:71–77.

^[6] Naugler WE, Karin M. NF-κB and cancer — identifying targets and mechanisms. Current Opinion in Genetics & Development. Feb 2008;18(1):19-26.

^[7] Peppicelli S, Bianchini F, Calorini L. Inflammatory cytokines induce vascular endothelial growth factor-C expression in melanoma-associated macrophages and stimulate melanoma lymph node metastasis. Oncol Lett. 2014;8(3):1133-1138. doi:10.3892/ol.2014.2297

^[8] Maloney JP, Gao L. Proinflammatory Cytokines Increase Vascular Endothelial Growth Factor Expression in Alveolar Epithelial Cells. Mediators of Inflammation. Sep 2015; https://doi.org/10.1155/2015/387842

^[9] Eibl G, Bruemmer D, Okada Y, Duffy JP, Law RE, Reber HA, et al. PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells. Biochem Biophys Res Commun. 2003;306:887–897.

^[10] Gately S. The contributions of cyclooxygenase-2 to tumor angiogenesis. Cancer Metastasis Rev. 2000;19:19–27.

^[11] Lee SH, Jeong D, Han YS, Baek MJ. Pivotal role of vascular endothelial growth factor pathway in tumor angiogenesis. Ann Surg Treat Res. 2015;89(1):1-8. doi:10.4174/astr.2015.89.1.1

^[12] Zhao Y, Adjei AA. Targeting Angiogenesis in Cancer Therapy: Moving Beyond Vascular Endothelial Growth Factor. Oncologist. 2015;20(6):660-673. doi:10.1634/theoncologist.2014-0465

^[13] Rak J, Yu JL, Kerbel RS, Coomber BL. What do oncogenic mutations have to do with angiogenesis/vascular dependence of tumors? Cancer Res. 2002;62:1931–1934.

^[14] Gopinathan G, Milagre C, Pearce OM, et al. Interleukin-6 Stimulates Defective Angiogenesis. Cancer Research. Aug 2015;75(15):3098-3107. DOI: 10.1158/0008-5472.can-15-1227.

^[15] Onnis B, Rapisarda A, Melillo G. Development of HIF-1 inhibitors for cancer therapy. J Cell Mol Med. 2009 Sep;13(9A):2780-6. doi: 10.1111/j.1582-4934.2009.00876.x. Epub 2009 Aug 8. PMID: 19674190; PMCID: PMC2832082.

^[16] Rapisarda A, Zalek J, Hollingshead M, et al. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res. 2004;64:6845–8.

^[17] Goldberg MA, Schneider TJ (1994) Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 269: 4355–4359.

^[18] Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, et al. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16: 4604–4613.

^[19]  Kim YJ, Lee HJ, Kim TM, Eisinger-Mathason TS, Zhang AY, Schmidt B, Karl DL, Nakazawa MS, Park PJ, Simon MC, Yoon SS. Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1alpha. Int J Cancer. 2013;132: 29-41.

^[20] Albertsson P, Lennernäs B, Norrby K. Low-dosage metronomic chemotherapy and angiogenesis: topoisomerase inhibitors irinotecan and mitoxantrone stimulate VEGF-A-mediated angiogenesis. APMIS. 2012;120(2):147-156. doi:10.1111/j.1600-0463.2011.02830.x

^[21] Salem ML, Kadima AN, El-Naggar SA, et al. Defining the ability of cyclophosphamide preconditioning to enhance the anti- gen-specific CD81 T-cell response to peptide vaccination: Creation of a beneficial host microenvironment involving type I IFNs and myeloid cells. J Immunother. 2007;30:40–53.

^[22] Matar P, Rozados VR, Gonzalez AD, et al. Mechanism of antimetastatic immunopotentiation by low-dose cyclophosph- amide. Eur J Cancer 2000;36:1060–1066.

^[23] Kono K, Kawaida H, Takahashi A, Sugai H, Mimura K, Miyagawa N, Omata H, Fujii H. CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers. Cancer Immunol Immunother. 2006;55:1064–1071.

^[24] Burton JH, Mitchell L, Thamm DH, Dow SW, Biller BJ. Low‐Dose Cyclophosphamide Selectively Decreases Regulatory T Cells and Inhibits Angiogenesis in Dogs with Soft Tissue Sarcoma. Journal of Veterinary Internal Medicine. July 2011. https://doi.org/10.1111/j.1939-1676.2011.0753.x

^[25] Torimura T, Iwamoto H, Nakamura T, et al. Metronomic chemotherapy: possible clinical application in advanced hepatocellular carcinoma. Transl Oncol. 2013;6(5):511-519. Published 2013 Oct 1. doi:10.1593/tlo.13481

^[26] Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux S, Martin F, et al. Metronomic cyclophosphamide regimen selectively depletes CD4 + CD25 + regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother. 2007;56:641–648. doi: 10.1007/s00262-006-0225-8.

^[27] Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, et al. CD4 + CD25 + regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–344. doi: 10.1002/eji.200324181.

^[28] Chen CA, Ho CM, Chang MC, Syu MH, Hsieh CY, Chen WF. Metronomic Chemotherapy Enhances Antitumor Effects of Cancer Vaccine by Depleting Regulatory T Lymphocytes and Inhibiting Tumor Angiogenesis. Molecular Therapy. June 2010;18(6):1233-1243.

^[29] Bellanti JA. (2012). Immunology IV: Clinical Applications in Health and Disease (4th edition). I Care Press.

^[30] Banissi C, Ghiringhelli F, Chen L, Carpentier AF. Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model. Cancer Immunol Immunother. 2009 Oct;58(10):1627-34. doi: 10.1007/s00262-009-0671-1. Epub 2009 Feb 17. PMID: 19221744.

^[31] Palmer DH, Midgley RS, Mirza N, Torr EE, Ahmed F, Steele JC, et al. A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma. Hepatology. 2009;49:124–132. doi: 10.1002/hep.22626.

^[32] Opzoomer JW, Sosnowska D, Anstee JE, Spicer JF, Arnold JN. Cytotoxic Chemotherapy as an Immune Stimulus: A Molecular Perspective on Turning Up the Immunological Heat on Cancer. Front. Immunol. Jul 2019. https://doi.org/10.3389/fimmu.2019.01654

^[33] Chan TS, Hsu CC, Pai VC, Liao WY, Huang SS, Tan KT, et al. Metronomic chemotherapy prevents therapy-induced stromal activation and induction of tumor-initiating cells. J Exp Med. 2016;213:2967–88. doi: 10.1084/jem.20151665

^[34] Neophytou CM, Pierides C, Christodoulou M, Costeas P, Kyriakou T, Papageorgis P. The Role of Tumor-Associated Myeloid Cells in Modulating Cancer Therapy. Front. Oncol. Jun 2020. https://doi.org/10.3389/fonc.2020.00899

^[35] Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 2006;6:295–307. doi: 10.1038/nri1806

^[36] Gong, D., Shi, W., Yi, S. et al. TGFβ signaling plays a critical role in promoting alternative macrophage activation. BMC Immunol. 2012;13(31). https://doi.org/10.1186/1471-2172-13-31

^[37] Soares KC, Rucki AA, Kim V, et al. TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner. Oncotarget. 2015;6(40):43005-43015. doi:10.18632/oncotarget.5656

^[38] Chalmin F, Mignot G, Bruchard M, Chevriaux A, Vegran F, Hichami A, Ladoire S, Derangere V, Vincent J, Masson D, Robson SC, Eberl G, Pallandre JR, et al. Stat3 and Gfi-1 transcription factors control Th17 cell immunosuppressive activity via the regulation of ectonucleotidase expression. Immunity. 2012;36:362–373.

^[39] Krupinski J, Kumar P, Kumar S, Kaluza J. Increased expression of TGF-beta 1 in brain tissue after ischemic stroke in humans. Stroke; A Journal of Cerebral Circulation. 1996;27(5):852–7.

^[40] Dissen GA, Lara HE, Fahrenbach WH, Costa ME, Ojeda SR. Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression. Endocrinology. 1994;134(3):1146–54.

^[41] Mazzocca A, Fransvea E, Lavezzari G, Antonaci S, Giannelli G. Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation. Hepatology. 2009;50(4):1140–51.

^[42] Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, et al. Targeting the TGFbeta pathway for cancer therapy. Pharmacology & Therapeutics. 2015;147:22–31.

^[43] Akbari A, Amanpour S, Muhammadnejad S, Ghahremani MH, Ghaffari SH, Dehpour AR, et al. Evaluation of antitumor activity of a TGF-beta receptor I inhibitor (SD-208) on human colon adenocarcinoma. Daru: Journal of Faculty of Pharmacy, Tehran University of Medical Sciences. 2014;22:47.

^[44] Zhang M, Kleber S, Rohrich M, Timke C, Han N, Tuettenberg J, et al. Blockade of TGF-beta signaling by the TGFbetaR-I kinase inhibitor LY2109761 enhances radiation response and prolongs survival in glioblastoma. Cancer Research. 2011;71(23):7155–67.

^[45] K. Bryniarski, M. Szczepanik, M. Ptak, M. Zemelka, W. Ptak, Influence of

cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in

mice, Pharmacol Rep. 61 (2009) 550-557.

^[46] Zhang YH, He M, Wang Y, Liao AH. Modulators of the Balance between M1 and M2 Macrophages during Pregnancy. Front. Immunol. Feb 2017. https://doi.org/10.3389/fimmu.2017.00120

^[47] Gratchev A. TGF-β signalling in tumor associated macrophages. Immunobiology. 2017;222(1):75–81. doi:10.1016/j.imbio.2015.11.016

^[48] Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, et al. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci U S A. 1987; 84(16):5788–92. doi:10.1073/pnas.84.16.5788

^[49] Dijkgraaf EM, Heusinkveld M, Tummers B, Vogelpoel LTC, Goedemans R, Jha V, Nortier JWR, Welters MJP, Kroep JR, van der Burg SH. Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment. Cancer Res. April 2013;73(8):2480-2492; DOI: 10.1158/0008-5472.CAN-12-3542

^[50] Mantovani A, Allavena P. The interaction of anticancer therapies with tumor-associated macrophages. J Exp Med. 2015;212(4):435-445. doi:10.1084/jem.20150295

^[51] Alishekevitz D, Gingis-Velitski S, Kaidar-Person O, et al. Macrophage-Induced Lymphangiogenesis and Metastasis following Paclitaxel Chemotherapy Is Regulated by VEGFR3. Cell Rep. 2016;17(5):1344-1356. doi:10.1016/j.celrep.2016.09.083

^[52] Liu, Z., Kuang, W., Zhou, Q., & Zhang, Y. TGF-β1 secreted by M2 phenotype macrophages enhances the stemness and migration of glioma cells via the SMAD2/3 signalling pathway. International Journal of Molecular Medicine. 2018;42:3395-3403. https://doi.org/10.3892/ijmm.2018.3923

^[53] Karagiannis GS, Condeelis JS, Oktay MH. Chemotherapy-Induced Metastasis: Molecular Mechanisms, Clinical Manifestations, Therapeutic Interventions. Cancer Res. 2019;79(18):4567-4576. doi:10.1158/0008-5472.CAN-19-1147

^[54] Bertolini F, Paul S, Mancuso P, Monestiroli S, Gobbi A, Shaked Y, Kerbel RS. Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells. Cancer Res. 2003;63:4342–4346.

^[55] Ma Y, Shurin GV, Gutkin DW, Shurin MR. Tumor associated regulatory dendritic cells. Semin Cancer Biol. 2012;22:298–306. doi: 10.1016/j.semcancer.2012.02.010.

^[56] Shurin GV, Ma Y, Shurin MR. Immunosuppressive mechanisms of regulatory dendritic cells in cancer. Cancer Microenviron. 2013;6:159–167. doi: 10.1007/s12307-013-0133-3.

^[57] Shurin GV, Ouellette CE, Shurin MR. Regulatory dendritic cells in the tumor immunoenvironment. Cancer Immunol Immunother. 2012;61:223–230. doi: 10.1007/s00262-011-1138-8.

^[58] Bocci G, Francia G, Man S, et al. Thrombospondin 1, a me- diator of the antiangiogenic effects of low-dose metronomic chemotherapy. Proc Natl Acad Sci USA. 2003;100:12917–12922.

^[59] Folkman J. Endogenous angiogenesis inhibitors. APMIS. 2004;112:496–507.

^[60] Kamat AA, Kim TJ, Landen CN Jr., et al. Metronomic che- motherapy enhances the efficacy of antivascular therapy in ovarian cancer. Cancer Res. 2007;67:281–288.