Bystander elimination of antigen loss variants in established tumors

Cancers express antigens that are targets for specific cytotoxic T lymphocytes (CTLs). However, cancer cells are genetically unstable. Consequently, sub-populations of cancer cells that no longer express the target antigen may escape destruction by CTLs and grow progressively. We show that cytotoxic T cells indirectly eliminate these antigen loss variants (ALVs) in a model system when the parental cancer cells express sufficient antigen to be effectively cross-presented by the tumor stroma. When the parental tumor expressed lower levels of antigen, cytotoxic T cells eradicated the antigen-positive parental cancer cells, but the ALVs escaped, grew and killed the host. By contrast, when the parental tumor expressed higher levels of antigen, cytotoxic T cells eradicated not only the parental cancer cells but also the ALVs. This 'bystander' elimination of ALVs required stromal cells expressing major histocompatibility complex (MHC) molecules capable of presenting the antigen, and occurred in tumors showing evidence of stromal destruction. ALVs were apparently eliminated indirectly when tumor-specific CTLs killed stromal cells that were cross-presenting antigen produced by and released from antigen-positive cancer cells. These results highlight the general importance of targeting the tumor stroma to prevent the escape of variant cancer cells.     

Immune-suppressive properties of the tumor microenvironment

Solid tumors are more than an accumulation of cancer cells. Indeed, cancerous cells create a permissive microenvironment by exploiting non-transformed host cells. Thus, solid tumors rather resemble abnormal organs composed of the cancerous cells itself and the stroma providing the supportive framework. The stroma can be divided into the extracellular matrix consisting of proteoglycans, hyaluronic acid, and fibrous proteins, as well as stromal cells including mesenchymal and immune cells; moreover, it contains various peptide factors and metabolites. Here, we will focus on immune-modulating capacities of the tumor microenvironment.     

Tumor-host interactions: the role of inflammation

It is well established that interactions between tumor cells and the host tissue stroma play a key role in determining whether and how any given solid malignancy will develop. In most cases, tumor cells hijack stromal cell functions for their own benefit and ultimately dictate the rules of engagement to the host tissue microenvironment. However, the contribution of the different stromal cell components to tumor growth remains to be clarified. Because most solid tumors are accompanied by a local inflammatory response, it has long been thought that inflammation and carcinogenesis are related. If formal proof that cancer can be initiated by inflammation in the absence of exogenous carcinogens is still lacking, there is abundant evidence that the inflammatory response can play a central role in modulating tumor growth and progression. This review will discuss some of the mechanisms whereby inflammation can both enhance and inhibit tumor growth.     

The autophagic tumor stroma model of cancer or “battery-operated tumor growth”

The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the “Autophagy Paradox”. We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth”. In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression, and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy, by the up-regulation of natural endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy, by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression, and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a “lethal” aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy, and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.     

Molecular aspects of stromal-parenchymal interactions in malignant neoplasms

Carcinomas are composed of parenchymal and stromal elements, and the malignant behavior is principally dictated by the cancer cells. However, the malignant tumors not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Thus, the tumor stroma is significantly different from that of the neighboring tissues. Cancer cells may alter their stroma by cell-to-cell contact, soluble factors or by modification of the extracellular matrix (ECM), they induce myofibroblast differentiation and govern the desmoplastic stroma reaction. On the other hand, the stromal cells (especially the myofibroblasts) are able to modify the phenotype, invasiveness, metastatic capacity of carcinomas, typically promoting the progression. Regarding pancreatic cancer, the pancreatic stellate cells (PSCs) seem to be the key elements in the cross-talk between the parenchymal cells and the desmoplastic stroma. The tumor stroma is also rich in tumor-associated macrophages (TAM), but their role in the malignant process is contradictory and may be different in various tumor types, but most studies suggest a negative impact on the tumor growth. The relationship between the parenchymal and stromal elements is highly complex, they mutually alter their characteristics. Because the neostroma of the carcinomas largely seems to promote the invasiveness of the malignant tumors, novel therapeutic strategies are being evaluated targeting the stromal elements, with some encouraging, but still fragmentary results.     

The autophagic tumor stroma model of cancer or “battery-operated tumor growth”: A simple solution to the autophagy paradox

The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the “Autophagy Paradox.” We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth.” In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy by the upregulation of natural, endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a “lethal” aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.Key words: caveolin-1, autophagy, cancer associated fibroblasts, hypoxia, mitophagy, oxidative stress, DNA damage, genomic instability, tumor stroma, wasting (cancer cachexia), Warburg effect     

Tumor stroma-associated antigens for anti-cancer immunotherapy

Immunotherapy has been widely investigated for its potential use in cancer therapy and it becomes more and more apparent that the selection of target antigens is essential for its efficacy. Indeed, limited clinical efficacy is partly due to immune evasion mechanisms of neoplastic cells, e.g. downregulation of expression or presentation of the respective antigens. Consequently, antigens contributing to tumor cell survival seem to be more suitable therapeutic targets. However, even such antigens may be subject to immune evasion due to impaired processing and cell surface expression. Since development and progression of tumors is not only dependent on cancer cells themselves but also on the active contribution of the stromal cells, e.g. by secreting growth supporting factors, enzymes degrading the extracellular matrix or angiogenic factors, the tumor stroma may also serve as a target for immune intervention. To this end several antigens have been identified which are induced or upregulated on the tumor stroma. Tumor stroma-associated antigens are characterized by an otherwise restricted expression pattern, particularly with respect to differentiated tissues, and they have been successfully targeted by passive and active immunotherapy in preclinical models. Moreover, some of these strategies have already been translated into clinical trials.     

Fibrosis and cancer: A strained relationship

Tumors are characterized by extracellular matrix (ECM) deposition, remodeling, and cross-linking that drive fibrosis to stiffen the stroma and promote malignancy. The stiffened stroma enhances tumor cell growth, survival and migration and drives a mesenchymal transition. A stiff ECM also induces angiogenesis, hypoxia and compromises anti-tumor immunity. Not surprisingly, tumor aggression and poor patient prognosis correlate with degree of tissue fibrosis and level of stromal stiffness. In this review, we discuss the reciprocal interplay between tumor cells, cancer associated fibroblasts (CAF), immune cells and ECM stiffness in malignant transformation and cancer aggression. We discuss CAF heterogeneity and describe its impact on tumor development and aggression focusing on the role of CAFs in engineering the fibrotic tumor stroma and tuning tumor cell tension and modulating the immune response. To illustrate the role of mechanoreciprocity in tumor evolution we summarize data from breast cancer and pancreatic ductal carcinoma (PDAC) studies, and finish by discussing emerging anti-fibrotic strategies aimed at treating cancer.     

Targeted destruction of the orchestration of the pancreatic stroma and tumor cells in pancreatic cancer cases: Molecular basis for therapeutic implications

Pancreatic cancer is one of the most lethal malignancies, with a prominent desmoplastic reaction as its defining hallmark. The past several decades have seen dramatic progress in understanding of pancreatic cancer pathogenesis, including identification of precursor lesions, sequential transformation from normal pancreatic tissue to invasive pancreatic cancer and corresponding signature genetic events, and the biological impact of these events on malignant behavior. However, the currently used therapeutic strategies for epithelial tumor cells, which have exhibited potent antitumor activity in cell culture and animal models, have failed to produce significant effects in the clinic. The desmoplastic stroma surrounding pancreatic cancer cells, which accounts for about 90% of a tumor's mass, clearly is not a passive scaffold for cancer cells but an active contributor to carcinogenesis. Improved understanding of the dynamic interaction between cancer cells and the stroma will be important to designing effective therapeutic strategies for pancreatic cancer. This review focuses on the origin of stromal molecular and cellular components in pancreatic tumors, their biological effects on pancreatic cancer cells, and the orchestration of these two components.     

The advantages of co-culture over mono cell culture in simulating in vivo environment

Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.     

Notch1 Pathway Activity Determines the Regulatory Role of Cancer-Associated Fibroblasts in Melanoma Growth and Invasion

Cancer-associated fibroblasts (CAF) play a crucial role in regulating cancer progression, yet the molecular determinant that governs the tumor regulatory role of CAF remains unknown. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in CAF. CAF carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of CAF in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors.     

Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.     

Arousal of cancer-associated stromal fibroblasts: Palladin-activated fibroblasts promote tumor invasion

Cancer-associated fibroblasts (CAF), comprised of activated fibroblasts or myofibroblasts, are found in stroma surrounding solid tumors; these myofibroblasts promote invasion and metastasis of cancer cells. Activation of stromal fibroblasts into myofibroblasts is induced by expression of cystoskeleton protein, palladin, at early stages in tumorigenesis and increases with neoplastic progression. Expression of palladin in fibroblasts is triggered by paracrine signaling from adjacent k-ras-expressing epithelial cells. Three-dimensional co-cultures of palladin-expressing fibroblasts and pancreatic cancer cells reveals that the activated fibroblasts lead the invasion by creating tunnels through the extracellular matrix through which the cancer cells follow. Invasive tunneling occurs as a result of the development of invadopodia-like cellular protrusions in the palladin-activated fibroblasts and the addition of a wounding/inflammatory trigger. Abrogation of palladin reduces the invasive capacity of these cells. CAF also play a role in cancer resistance and immuno-privilege, making the targeting of activators of these cells of interest for oncologists.     

Arousal of cancer-associated stromal fibroblasts

Cancer-associated fibroblasts (CAF), comprised of activated fibroblasts or myofibroblasts, are found in stroma surrounding solid tumors; these myofibroblasts promote invasion and metastasis of cancer cells. Activation of stromal fibroblasts into myofibroblasts is induced by expression of cystoskeleton protein, palladin, at early stages in tumorigenesis and increases with neoplastic progression. Expression of palladin in fibroblasts is triggered by paracrine signaling from adjacent k-ras-expressing epithelial cells. Three-dimensional co-cultures of palladin-expressing fibroblasts and pancreatic cancer cells reveals that the activated fibroblasts lead the invasion by creating tunnels through the extracellular matrix through which the cancer cells follow. Invasive tunneling occurs as a result of the development of invadopodia-like cellular protrusions in the palladin-activated fibroblasts and the addition of a wounding/inflammatory trigger. Abrogation of palladin reduces the invasive capacity of these cells. CAF also play a role in cancer resistance and immuno-privilege, making the targeting of activators of these cells of interest for oncologists.     

Engineering tumor stromal mechanics for improved T cell therapy

Adoptive cellular therapies (ACT), including the engineered T cell receptor (TCR) therapy and chimeric antigen receptor (CAR) T Cell Therapy, are currently at the forefront of cancer immunotherapy. However, their efficacy for the treatment of solid tumors has not been confirmed. The fibrotic stroma surrounding the solid tumor has been suggested as the main barrier in the disarmament and suppression of the engineered T cells. In this review, we will discuss the recent findings on the mechanism of T cell suppression by the tumor stroma with a special emphasis on the effect of stromal mechanics. We will also discuss the engineering approaches used to dissect the mechanism of the T cell suppression by the stromal mechanical factors. Finally, we will provide a future outlook on the strategies to improve the efficacy of T cell therapy through altering the tumor stromal fibrosis.     

Energy transfer in "parasitic" cancer metabolism

It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, “parasitic” energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a “parasitic organism.” Hence, we propose the term “Parasitic Cancer Metabolism” to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can confer drug resistance. Importantly, this metabolic chemo-resistance is reversed by blocking OXPHOS in cancer cell mitochondria with drugs like Metformin, a mitochondrial “poison.” In summary, parasitic cancer metabolism is achieved architecturally by dividing tumor tissue into at least two well-defined opposing “metabolic compartments:” catabolic and anabolic.     

Fibroblast and prostate tumor cell cross-talk: Fibroblast differentiation, TGF-β, and extracellular matrix down-regulation

Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. The influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant down-regulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PC3 and DU145. Interestingly, TGF-β down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and α5β1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after cross-talk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with “activated” stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM.