Targeting Tumor Microenvironment in Pancreatic Cancer: A Translational Approach

Pancreatic cancer (PC), i.e., pancreatic ductal adenocarcinoma (PDAC), is one of the most aggressive and deadly cancers worldwide, it occupies position 7 among cancer-related causes of death, whereas its prognosis is not any better since the mortality rate of PDAC patients does not exceed 12%. The source of this high lethality is the later clinical manifestation of the disease, massive metastatic nature and massive resistance to chemotherapy, radiotherapy, and immunotherapy. Unlike most other solid neoplasms, PDAC has a characteristic histopathological structure, in which a dense network of desmoplastic stroma represents one of the key components of tumor microenvironment (TME) [1].

The pancreatic TME is a complex dynamic environment that includes cancerassociated fibroblasts (CAFs), pancreatic stellate cells (PSCs), immune suppressive myeloid and lymphoid cells, extracellular matrix (ECM) components and abnormal vascular network. It does not only provide structural scaffolding, but also actively promotes tumour progression by promoting fibrosis and inhibition of immune mediated tumor elimination by inflammation. This unregulated deposition of these ECM and a hyper activation of the stromal components create a physical and bio chemo barrier preventing good delivery of drugs and providing therapeutic resistance[2].

Recent data suggests that TME-malignant cell interactions are a two-way process that is vital in tumor maintenance. Therefore, the regulation of stromal and immune TME constituents has become one of the potential therapeutic targets. Translational approaches, which are intended to moderate TME (depletion of stromal cells, remodeling of ECM, and immune reprogramming), are even intended to enhance drug delivery as well as sensitize tumors to traditional and neo-targeted therapies, with the goal of improving the curative outlook of this potential otherwise inexorable disease[3].

2. Tumor Microenvironment in Pancreatic Cancer

Tumor microenvironment (TME) of pancreatic ductal adenocarcinoma comprises a highly complex environment, both cellular and acellular and constitutes dynamic interactions between it and malignant cells. The complex system has an extensive effect on tumourigenesis, metastatic spread and resistance to therapy, making it an essential hub of interest in oncology studies[4].The stromal compartment is one of the hallmarks of the pancreatic TME. Cancer-related fibroblasts (CAFs), and pancreatic stellate cells (PSCs) become the main stromal components, which are responsible for the large-scale extracellular matrix (ECM) production[5]. The resultant effect of their activation is fibrotic remodelling and increase of tumour stiffness, which hamper the delivery of the chemotherapeutic agents to the tissues through the vascular system. This reciprocal crosstalk between the stromal cells and the cancer cells enhances tumour proliferation, invasion and provides a permissive environment that supports invasion by metastasis[6].

The pancreatic tumour microenvironment is defined by an excessive condition of immunosuppression. It is mostly colonized by tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) as well as by regulatory T cells (Tregs). Cytotoxic T -cell activity is blocked by these populations to reduce antitumour immunity and promote immune tolerance. As a result, pancreatic tumours often avoid immune surveillance and that is why it is one of the reasons the modern immunotherapeutic approaches have limited effect on this malignancy[7].Pancreatic tumour vascular characteristics are characterized by being hypovascular and disorganized. Poor perfusion will create hypoxic niches which precondition tumour aggressiveness and reprogramming of metabolism. Hypoxic microenvironment also increases angiogenic, epithelial 0 -mesenchymal transition and resistance against conventional modalities signalling cascades[8].The extracellular matrix compounds therapeutic issues by having a dense and fibrotic structure. It is not only a mechanical barrier to diffusions of drugs, but also a biological framework that induces cellular motility and invasion[9]. The complex interaction of stromal, immune, vascular, and matrix makes up an unfavorable but conducive environment which drives the progression of pancreatic cancer. To this end, selective manipulation of the TME has a significant potential in improving therapeutic outcome against this very deadly ailment[10].

3. Cellular and Molecular tumor microenvironment Players in Pancreatic Cancer.

PDAC Tumor microenvironment (TME) is an extremely complex but dynamic system composed of various components of cells and molecules that have complicated interactions with malignant cells. Such interactions affect tumor development, resistance to treatment, and finally clinical results are achieved. To develop specific therapeutic measures, it is necessary to have a detailed knowledge of the cellular and molecular components of the TME.Cellular elements of the Pancreatic TME are given below[11].

3.1Fibroblasts in cancer and pathophysiology: Cancer-Associated Fibroblasts (CAFs): 

CAFs are one of the richest stromal cell types in PDAC and contribute to the prominent fibrotic reaction witnessed in this cancer type. They produce structural proteins advancing growth (collagen and fibronectin) as well as cytokines and growth factors that maintain the growth of the tumor[12]. CAFs are heterogenous, and they contain different subpopulations that have different functional roles. Some of the subtypes facilitate tumor progression and invasion and immune suppression whereas others can restrain tumor growth by imposing mechanical support or paracrine signaling. This duality has some therapeutic prospects and challenges[13].

3.2 Independent cell types: Pancreatic Stellate Cells (PSCs): 

PSCs in their dormant condition preserve a normal pancreatic structure. When stimulated by inflammatory stimuli or tumor-associated stimuli, they become myofibroblast-like cells, which hyperproduce extracellular matrix (ECM) components causing the phenomenon of desmoplasia a characteristic of PDAC[14]. The process makes tissues stiffer and vascularization inhibited. PSCs also secrete growth factors and cytokines that promote tumor growth, migration as well as chemoresistance.Here, a distinctive alteration in the clinical efficacy of chemotherapy and radiotherapy has been proposed, resulting from the natural alteration of macrophages into Tumor-Associated Macrophages (TAMs).:  TAMs present a significant immune part of the TME and are concentrated towards the M2 type which favours tumorgrowth[15]. M2 macrophages release the pro-angiogenic and immunosuppressive determining factors that enhance metastasis and suppress good T- cells reaction. Poor prognosis is associated with a large number of TAMs and myocardial infarction, which is why they are important potential therapeutic targets[16].

3.3 Myeloid -Derived Suppressive Cells (MDSCs): 

MDSCs have a central role to play in suppressing antitumor immunity. They inhibit T-cell activation with the synthesis of arginase and reactive oxygen species and thus they regulate immune evasion by the tumor. Their proliferation in the microenvironment of the PDAC also complicates the strategies of immunotherapy[17].Endothelial Cells: No erythematous changes observed at `stage.|human|Vasculature: No erythematous changes seen at this point. The PDAC blood supply is characterized by a significant irregularity and inadequacy, which play a role in the development of hypoxia, acidosis, and drug delivery limitations[18]. Abnormal endothelial cues impart ineffective perfusion; consequently, this triggers adaptive survival mechanisms in the tumor cells, and it leads to aggressive characteristics and tolerance to chemotherapy and radiotherapy[19].

3.4 TME Molecular Mediators of TME Function.

Major molecular mediators coordinate the tumor cells with properties of TME. The factors that influence growth and activation of fibrosis, angiogenesis, and immune modulation involve transforming growth factor -b (TGF -b) and vascular endothelial growth factor (VEGF)[20]. The immunosuppressive milieu is further supported by the cytokines such as interleukin- 6(IL-6) and interleukin-10 (IL-10). There is an attraction of cancer cells to immune and stromal cells assisted by chemokines like CXCL12 which promotes the survival and proliferation of cancer cells. Additionally, matrix metalloproteinases (MMPs) reorganize the ECM simulating the invasion and the biochemical environment of the TME[21].

The interaction between CAFs, PSCs, immune cells, endothelial elements, and a sophisticated molecular network in their complex interplay is the basis of the pancreatic TME. The understanding of these communications is also necessary in creating new, combination-based therapies to interfere with tumor stroma crosstalk and enhance patient outcomes in pancreatic cancer[22].

4. Targeting Tumor Microenvironment: Preclinical Advances

Attack on tumor microenvironment (TME) is an approach that has been developed as one of the promising options in overcoming therapeutic resistance and improving cancer treatment results. The thick extracellular matrix (ECM) of tumors is a mechanical barrier to drug delivery; as such strategies to overcome this has led to the development of stromal ablation therapies to counteract the barrier[23]. Hyaluronic acid can be degraded by hyaluronidase and PEGylated hyaluronic acid PEGPH20 in particular, which leads to a decrease in pressure in the interstitial fluid and an increase in vascular perfusion[24]. This remodelling enhances the effectiveness of chemotherapeutic agents by supporting the efficient delivery of chemotherapeutic agents. Likewise, the anti-fibrotic drugs like pirfenidone and losartam weaken fibrosis through suppressing the activation of fibroblasts and the deposition of collagen which normalizes the ECM and enhances the diffusion of drugs within tumour tissue[25].

Another important aspect of TME modulation is that of vascular normalization. Tumour vasculature has often been characterized by lack of structure, leakage and ineffectiveness, resulting in hypoxia and limited delivery of therapeutic factors. Transient restoration of vessel architecture and functioning with anti-angiogenic interventions in antagonism of the vascular endothelial growth factor (VEGF) signalling pathway[26]. This normalisation increases oxygen and nutrient penetration as well as facilitating drug penetration. Overall, though, those progresses in preclinical research of stromal depletion, anti-fibrotic treatment practices and vascular normalisation underline the importance of TME-targeted approaches in overcoming drug resistance and in enhancing the actions of traditional or new anti-cancer drugs[27].

4.1 Immune Microenvironment Modulation

Dynamics of the immune microenvironment are one of the key areas of study in regards to pancreatic ductal adenocarcinoma (PDAC) as the tumour exhibits an immunosuppressive environment that thus hinders effective antitumour responses.PDAC has shown limited response to checkpoint blockade agents targeting PD-1, PD-L1 and CTLA-4 but responses can be explained within the context of a low tumour mutational burden, insufficient tumour-immune-cell infiltration and a dense stromal compartment with a restraining ability on immune activation[28]. Modern research strives to enhance the treatment effect of these inhibitors with a combination of chemotherapy, radiotherapy, or stromal-directed agent.  Examples of cancer vaccines under development include GVAX and CRS 207 used as remodelling agents of the tumour microenvironment and inducing potent immune responses[29]. GVAX uses irradiated tumour cells that express granulocyte -macrophage colony -stimulating factor (GM -CSF) to enhance antigen presentation and T -cell activation, and CRS -207 uses a live attenuated -Listeria monocytogenes -vector that expresses mesothelin in order to enhance cytotoxic T -cell activity[30]. 

The overall goal of these interventions is to first convert the immunologically cold PDAC tumour into a hot phenotype by refilling and mediating effector T cells.  Among other adoptive cell therapies, which are of current preclinical and clinical interest, are chimeric antigen receptor (CAR) -T cells and tumour-infiltrating lymphocyte (TIL) Strategies. These modalities have so far shown moderate efficacy, but when combined with tumour stroma remodelling or immune-cells trafficking agents, show promise and revitalizes hope of immunotherapeutic efforts in PDAC[31].

4.2 Nanomedicine Approaches

Nanomedicine could be used to powerfully address the stromal and vascular barriers of tumor microenvironment (TME) to increase therapeutic delivery and effectiveness. Nanoparticles may be designed in a manner to enhance the diffusion of anticancer drugs into the thickened extracellular matrix, and targeted drug release at the tumorlocation[32]. These nanoscale systems allow the controlled delivery of drugs, prolonged times of circulation and a decrease in off-target toxicity. It is also possible to prepare functionalized nanoparticles to react to a set of tumor-associated signals, including pH, enzymatic activity, or hypoxia to release their payload selectively in the TME[33]. Liposomes, polymeric nanoparticles and micelles are examples of nanocarriers which have been shown to be useful in the delivery of chemotherapeutic agents in pancreatic cancer and other desmoplastic tumors with minimal systemic side effects[34].

Table 1: Major Cellular and Molecular Components of TME in Pancreatic Cancer and Their Roles

5. Translatoral and Clinical Developments.

In the last ten years, translational oncology has made significant contributions to our understanding of the pancreatic tumor microenvironment (TME) and its impact on the therapeutic outcome. In pancreatic ductal adenocarcinoma (PDAC) desmoplasia, aberrant vascular architecture, and a strong level of immunosuppression characterize the TME and impede drug delivery into cancer cells and anti-tumor immune response[40]. Translational studies have attempted to be able to translate preclinical findings on stromal and vascular dynamics into clinical modalities that are able to overcome these barriers. Scientists have studied the relationship between tumor stroma, immune components and vascular integrity to come up with combination therapies that are able to attack the malignant cells and their supportive milieu[41]. These efforts balance both laboratory findings and clinical treatments thus leading to an integrative treatment paradigm. The subsequent clinical trials efforts have led to subjects of stromal modulation, immunotherapeutic treatments, and vascular normalization, as potentially effective approaches to improve survival and response to treatment in patients with PDAC[42].

 5.1 TME-Targeted Clinical Trials

The therapeutic use of TME-based approaches has produced numerous clinical trials which have tried to manipulate the stromal components, normalization of tumor vasculature, and the enhancement of the anti-tumor immunity to a greater extent[43]. Since PDAC is resistant to traditional cytotoxic therapy, TME-based component targeting has become an important adjunct therapy. Stromal depletion or remodeling trials instead of hyaluronidase-based enzymes have also tried to reduce tumoral interstitial pressure and increase the penetration of drugs. Similarly, the process of normalizing vascular phenotype is meant to restore effective perfusion and thus increasing chemotherapeutic potency and oxygen delivery[44]. The monotherapy of immune checkpoint blockade has not been associated with significant effect in PDAC because of immune exclusion of stroma mediated by TME; nonetheless, combinatorical studies of stroma- and vessel-modulating mediators are currently being pursued[45].

5.1.1 PEGPH20

PEGPH20 is a pegylated recombinant hyaluronidase-derived hyaluronan (HA) polymer, which is better positioned to break down the hyaluronic acid polymer (HA) that is also responsible in PDAC to raise intrastitial pressure and cause inadequate vascular perfusion. Initial pilot trials showed PEGPH20 used together with conventional chemotherapy agents, including nab -paclitaxel and gemcitabine, had a significant additive effect on progression-free survival in patients with HA -high tumors, suggesting a biomarker-based advantage. PEGPH20 breaks down HA, which leads to the physical barrier of the malignant cells being broken, allowing drugs to enter the tumor bed and oxygenation to occur more easily[46]. Later phase-III trials however gave mixed results with certain subpopulations of patients benefiting and others developing increased toxicity and thrombo-embolic events. The findings support the heterogeneity of the stromal targeting and the inherent heterogeneity of PDAC stroma[47].

5.1.2 Losartan

Angiotensin-II receptor antagonist Losartan, traditionally used as a hypertension drug, has been suggested to be used as a stromal-modifying and vascular-normalizing agent in PDAC. Its suggested mechanism involves the reduction of collagen and hyaluronan production by the cancer-related fibroblasts thus reducing the stiffness of the extracellular matrix and interstitial fluid pressure[48]. The effect of this remodeling increases perfusion as well as helps deliver chemotherapeutic agents better into the tumoral core. The preclinical models have demonstrated that losartan can be used in synergy with chemotherapy and radiotherapy by increasing the concentration of drugs and oxygen level intra tumor[49]. Losartan has also been tested in clinical practice with standard chemotherapy in the management of a locally advanced PDAC with promising results of response and resectability[50].

5.1.3 Checkpoint Blockade

Although immune checkpoint inhibitors (ICIs) have been shown to have a transformative success in multiple malignancies, their application in PDAC has had a low response. The TME associated with immunosuppressive reaction, which is characterized by the exclusion of cytotoxic T cells, infiltration of regulatory T cells, and preeminence of myeloid-derived suppressive cells make PDAC highly resistant to single-agent ICI therapy[51]. It has been explored in clinical trials that incorporation of ICIs with any of the previously listed therapies as well as oncolytic virus and stromal modulators can be used to enhance immune infiltration and activation[52]. The most promising ones are to combine PD-1 /PD-L1 blockers with agents that can interfere with the stromal barrier or normalise vasculature to allow an increase in immune cell penetration. On-going research is also focused on the identification of predictive biomarkers, including but not limited to microsatellite instability, tumor mutational burden, and immune signature to assist patient selection[53]. The attempts are a significant step to achieving effective immunotherapy of PDAC by redefining the immune contexture and neutralizing the suppressive effects of TME[54].

5.2 Combination Therapies.

Combinations of tumor microenvironment (TME)-targeted therapy with standard therapeutic systems such as chemotherapy, radiotherapy and immunotherapy have been shown to enhance antitumor properties in pancreatic ductal adenocarcinoma (PDAC). The presence of desmoplastic stroma, deviant vasculature, and immunosuppressive milieu that is typical of PDAC poses considerable challenges to drug delivery and immune infiltration, thus making monotherapies to be of little use[55]. With the use of TME-modulating drugs, these deterrents can be reduced and allow standard therapeutic regimens to increase their penetration and activity. Indicatively, stromal depletion through hyaluronan or collagen targeting agents reduces interstitial pressure which in turn increases perfusion of chemotherapeutic agents including gemcitabine and nab-paclitaxel[56]. Similarly, vascular normalization drugs like losartan enhance the tissue oxygenation and radiosensitivity, and hence maximize the outcomes of radiotherapies[57].

At the same time, mutually energizing immune checkpoint inhibitors with stroma-neutralizing or angiogenesis-neutralizing signs delivering a bombarding evidence of crucial case in preclinical efforts and preclinical studies. The aim of these regimens is to re-establish the inherently immunologically cold PDAC microenvironment to a more inflamed or hot phenotype which the immune can be activated[58]. In addition, the combination of cytotoxic and immunotherapeutic treatments allow inducing immunogenic cell death, which promotes long-term antitumor immune responses. The critical role of the strategic sequencing of therapeutic interventions is also present; pretreatment by stromal modifiers may create temporal windows, which increase the perfusion and immune accessibility[59].Altogether, the combination therapies to be used simultaneously to address the tumor mass and reestablish its microenvironment present a reasonable and promising approach to overcoming the intrinsic resistance to therapeutic agents in PDAC. Clinical studies are an ongoing process that is still polishing these combination regimens and there is a current emphasis on biomarker guided individualisation that aims at maximising the synergistic therapeutic effects and reducing the toxicity associated with treatment[60].

5.3 Biomarkers

Biomarkers have to be reliable to be able to refine the tumour microenvironment (TME)-targeted therapeutics and improve clinical outcomes in pancreatic ductal adenocarcinoma (PDAC). The inherent biological heterogeneity of PDAC, combined with its complexity of the microenvironment, does not allow a monolithic effect of the therapy in all patients[61]. This has led to the intensive studies of novel markers like circulating tumour DNA (ctDNA), stromal signatures and immune profiling to guide selection of therapy, as well as to monitor response to therapy and disease persistence in the aftermath of treatment.tDNA is a non-invasive real-time surrogate of tumour dynamics that can be used to predict resistance and disease progression, and also to detect residual disease after therapy. In addition, change in ctDNA amounts can predict treatment efficacy before radiologic changes appear[62]. 

Markers of the stroma and extracellular matrix (ECM) including the level of hyaluronan, collagen density, and fibroblast activation markers are under consideration as predictive markers of stroma responsiveness to stromal targeted agents such as PEGPH20 or losartan[63]. The patients reported to have strong stromal rigidity or hyaluronan-containing tumours can receive increased benefit of TME-modulating interventions. Moreover, immune profiling of TME, such as the counting of tumour-infiltrating lymphocytes, myeloid-derived suppressor cells, and cytokine expression patterns, can be used to determine patient subsets that might be hyper responsive to immunotherapy or a combination of immunotherapy[64].  The combination of these biomarker analyses supplements individualized treatment plans and allows the tailoring of treatment to be dynamic. With the potential for future trials that would prove these indicators, precision targeted TME presents a daunting future of revolutionizing the treatment of PDAC and therefore making it no longer a homogenous and one-size-fits-all paradigm, but rather a biomarker-driven, patient-centered treatment paradigm[65].

Table 2: Selected Clinical Trials Targeting TME in Pancreatic Cancer