Increased immune cell infiltration in patient-derived tumor explants treated with Traniplatin: an original Pt(iv) pro-drug based on Cisplatin and Tranilast

Abstract

An expanding body of contemporary scientific research has consistently highlighted a profound and increasingly recognized correlation between the presence of an elevated immune cell infiltrate within the tumor microenvironment and a significantly improved prognosis for patients afflicted with cancers originating from diverse tissue types. This paradigm shift in understanding cancer biology underscores the critical role played by the host’s immune system in combating malignant progression and represents a cornerstone of the burgeoning field of immuno-oncology. An improved prognosis in this context generally translates to more favorable patient outcomes, including enhanced overall survival rates, reduced disease recurrence, and a better quality of life post-treatment, observed across various cancers such as colorectal carcinoma and melanoma.

In light of this evolving understanding, a novel therapeutic agent, Traniplatin (TPT), has emerged as a promising candidate. TPT is ingeniously designed as a platinum(IV) pro-drug, building upon the established chemotherapeutic agent Cisplatin (CDDP) and incorporating elements from the already marketed pharmaceutical compound, Tranilast. The design as a platinum(IV) pro-drug is particularly significant, as it often confers advantages such as enhanced stability, reduced reactivity in systemic circulation, and a more controlled release of the active platinum species specifically within the tumor milieu, potentially leading to improved efficacy and reduced systemic toxicity compared to its precursor, Cisplatin, which is known for its potent cytotoxic effects but also its significant side effects. The incorporation of Tranilast, a drug with known anti-allergic and anti-fibrotic properties, suggests a potential for multi-modal action or a specific targeting rationale in the design of TPT.

Initial comparative investigations conducted in carefully controlled *in vitro* settings revealed compelling differences in the cytotoxic profiles of TPT when juxtaposed with Cisplatin. Specifically, TPT demonstrated a remarkably increased cytotoxic activity against established colon and lung cancer cell lines, indicating its potent anti-tumor efficacy. Crucially, and perhaps more importantly given the contemporary emphasis on immune preservation in cancer therapy, TPT exhibited a significantly decreased cytotoxic activity against various immune cells. This differential selectivity is a highly desirable characteristic for a chemotherapeutic agent, as it suggests a reduced potential for inducing systemic immunosuppression, thereby potentially preserving the host’s endogenous anti-tumor immune responses, which are vital for long-term disease control and improved patient outcomes.

To further bridge the gap between *in vitro* observations and real-world clinical relevance, the therapeutic efficiency of TPT was subsequently rigorously evaluated in a more physiologically representative model: tumor explants directly derived from colorectal cancer samples. These invaluable samples were obtained from patients who had undergone intended curative surgery, providing a unique opportunity to assess the drug’s activity within a complex tumor architecture that closely mimics the patient’s biological environment.

The findings from these ex vivo studies were remarkably consistent with and further expanded upon the *in vitro* observations. TPT induced robust and undeniable intra-tumoral cytotoxic activity, unequivocally demonstrating its capacity to effectively eliminate cancer cells within patient-derived tissue. Intriguingly, this potent anti-cancer effect was concomitantly associated with an elevated presence of immune cell infiltrate within the treated tumor explants. This observation suggests that TPT not only effectively targets malignant cells but also possesses the remarkable ability to either spare the resident immune cells from cytotoxic damage or potentially even foster an increased accumulation of these critical immune components within the tumor microenvironment. This collective evidence strongly points towards a reduced cytotoxic activity of TPT specifically against immune cells within the complex context of colorectal cancer, reinforcing its potential as an immune-sparing or even immune-enhancing anti-cancer therapeutic, with promising implications for combination therapies and overall patient prognosis.

Introduction

Colorectal cancer, a malignancy originating in the colon or rectum, represents a formidable global health challenge, with its incidence and mortality rates continuing to pose a significant burden. Annually, approximately 1.4 million new cases are diagnosed worldwide, leading to nearly 700,000 deaths. This positions colorectal cancer as the third leading cause of morbidity and the fourth most frequent cause of cancer-related mortality across the globe. For patients diagnosed with stage II/III disease, and particularly for those with advanced stage IV metastatic disease, the current standard of care following intended curative surgery often involves a combination chemotherapy regimen, typically consisting of 5-fluorouracil in conjunction with oxaliplatin, a treatment regimen commonly known as FOLFOX. While such platinum-based treatments offer a tangible potential for improvement in patient outcomes, it is an unfortunate reality that a substantial number of patients either do not respond adequately to these therapies or experience only a marginal gain in overall survival. Given the current trajectories, projections indicate that the global incidence of colorectal cancer is expected to increase dramatically, potentially surpassing 2.2 million new cases annually, and associated deaths could rise to 1.1 million by the year 2030. This alarming forecast underscores the critical and urgent need for the development and successful translation of innovative and more effective therapeutic strategies into the clinical setting.

Cisplatin, commonly abbreviated as CDDP, is a well-established and widely utilized chemotherapeutic agent that has received approval from the US Food and Drug Administration (FDA). It is routinely employed in the treatment of various cancers originating from distinct tissues, including but not limited to colon, testicular, ovarian, lung, and head and neck cancers. Despite its proven efficacy in combating malignant diseases, the inherent benefits of platinum-based therapies like Cisplatin are frequently counterbalanced by the emergence of severe and often dose-limiting side effects. These adverse reactions can include nephrotoxicity, a damaging effect on the kidneys; myelotoxicity, which suppresses bone marrow activity and blood cell production; neurotoxicity, affecting the nervous system; and highly distressing symptoms such as severe vomiting and nausea. The occurrence and severity of these side effects often critically impede or even prevent the completion of the intended treatment course, thereby compromising therapeutic outcomes. Consequently, considerable scientific attention has been directed towards the strategic development of more stable platinum(IV) complexes. These novel compounds are specifically engineered to mitigate some of the aforementioned limitations inherent to existing platinum(II)-based drugs. Platinum(IV) species are increasingly recognized as highly promising anti-cancer agents, primarily because they offer enhanced stability compared to their corresponding platinum(II) species, a characteristic attributed to their d6 octahedral geometry versus the d8 square planar geometry of Pt(II) complexes. Furthermore, platinum(IV) complexes possess the advantageous property of being able to navigate to the cancerous cellular environment without extensively reacting with sulfur-containing biomolecules. These biomolecules are widely understood to be a primary contributor to the nephrotoxicity induced by platinum(II) analogues. A compelling example of such a promising development is Satraplatin, an orally administered platinum(IV) complex that successfully advanced to phase III clinical trials, highlighting the clinical potential of this class of compounds. The fundamental mechanism of action for platinum(IV) pro-drugs involves their intracellular reduction to the corresponding, biologically active platinum(II) species. This reduction process is crucial for the drug’s anti-proliferative activity, and it is accompanied by the concomitant release of their axial ligands. The occurrence of this intracellular reduction has been rigorously demonstrated and confirmed through advanced analytical techniques such as XANES spectroscopy and fluorescence methods. It has also been extensively reported that the physicochemical properties of platinum(IV) complexes, including their aqueous solubility or lipophilicity, can be precisely tuned through relatively simple modifications to their axial substituents, offering a powerful avenue for optimizing drug delivery and efficacy.

Within this framework, Tranilast emerges as an axial ligand of particular interest. Tranilast is a small molecule that has been clinically commercialized in Japan and Korea as a well-established anti-allergic drug, primarily used for the treatment of various inflammatory diseases, including bronchial asthma, allergic conjunctivitis, atypical dermatitis, and for the management of keloids and hypertrophic scars, highlighting its low toxicity profile. Structurally, Tranilast is an analogue of a tryptophan metabolite. Beyond its anti-allergic properties, Tranilast has also been shown to reduce pathological fibrosis associated with conditions like myocardial infarction and to inhibit the migration of human breast cancer cells. More recently, Tranilast has garnered significant attention for its distinct properties as a cytostatic agent, demonstrating efficacy in inhibiting the growth of prostate, breast, and pancreatic cancer cells, as well as in gliomas and various other solid tumors. Furthermore, its extensive application in the clinical setting over several years has unequivocally demonstrated that Tranilast is a relatively safe drug, presenting only modest and generally well-tolerated side effects, even at daily doses of 600 milligrams administered over periods of several months. The precise molecular mechanism by which Tranilast exerts its anti-proliferative effects remains a subject of ongoing scientific debate. Some evidence suggests that it acts by suppressing the transforming growth factor-β (TGF-β) pathway, a critical regulator of cell growth, differentiation, and extracellular matrix production. However, other molecular targets have been proposed, including the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-9 production, which are enzymes involved in tissue remodeling and cancer metastasis; the inhibition of epithelial-mesenchymal transition (EMT), a process crucial for cancer invasion; and the suppression of the activation of key intracellular signaling pathways such as nuclear factor kappa B (NF-kB), protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). Building upon this extensive background, the present communication reports the innovative synthesis, comprehensive characterization, and in-depth biological evaluation of Traniplatin (TPT), a novel platinum(IV) pro-drug featuring Tranilast strategically incorporated in its axial position, aiming to leverage the combined beneficial properties of both components for enhanced anti-cancer therapy.

Materials And Methods

The synthesis of the novel platinum(IV) derivative, Traniplatin (TPT), involved the successful conjugation of Tranilast to Cisplatin. This was achieved through a chemical reaction between oxoplatin and the N-hydroxysuccinimide (NHS)-ester derivative of Tranilast. Following its synthesis, the new TPT compound underwent rigorous characterization to confirm its identity and purity. This comprehensive characterization utilized a suite of analytical techniques, including elemental analyses, nuclear magnetic resonance (NMR) spectroscopy for both hydrogen-1, carbon-13, and platinum-195 nuclei, and high-performance liquid chromatography (HPLC). It is important to note that attempts to synthesize a bis-adduct, a derivative with two Tranilast ligands, were unsuccessful under the employed conditions. Given that the mechanism of action of platinum(IV) pro-drugs fundamentally relies on their intracellular reduction to the corresponding platinum(II) species, which are responsible for the therapeutic anti-proliferative activity and the concomitant release of axial ligands, the reduction kinetics of TPT were thoroughly investigated. This reduction process was studied via HPLC, demonstrating that TPT was completely reduced by ascorbic acid, leading to the quantitative release of Tranilast within 30 hours. Furthermore, stability studies confirmed that TPT remained stable for up to 24 hours in an aqueous/acetonitrile solution, ensuring its integrity under physiological conditions relevant for administration.

The biological activity of the newly synthesized TPT was extensively evaluated across a panel of diverse human-originating cancer cell lines. These included prostate cancer cell lines (PC-3 and C4-2), cervical cancer cells (Hela), colorectal cancer cells (HT29), and lung cancer cells (A549), providing a broad assessment of its cytotoxic potential. TPT’s efficacy was systematically compared to that of Cisplatin, free Tranilast, and a 1:1 mixture of Cisplatin and Tranilast. Initial assessments in prostate and cervical cancer cells demonstrated that TPT exhibited cytotoxic activity within a range comparable to that of Cisplatin, suggesting general anti-cancer properties. More notably, and of particular interest for its therapeutic implications, TPT was found to be approximately 4-fold more potent than Cisplatin in lung cancer cells and a remarkable 10 times more cytotoxic than Cisplatin against colorectal cancer cells. This differential potency strongly suggested a specific and enhanced affinity of TPT for lung and colon cancer cells, indicating a potential for targeted efficacy. It was also observed that the free axial ligand, Tranilast, demonstrated only weak cytotoxic activity against all tested cancer cell lines under the experimental conditions employed, underscoring that the potent effects observed with TPT were primarily attributable to the platinum complex rather than the isolated ligand.

Beyond merely assessing cytotoxicity, the study next analyzed TPT’s capacity to induce programmed cancer cell death. To achieve this, the activation of caspase-3/7, an essential enzymatic step in the apoptotic cascade, was precisely measured in treated cancer cells. Optimal experimental settings were meticulously defined for each specific cancer cell line to ensure accurate assessment. The results showed that incubation of both lung cancer cells (A549) and colon cancer cells (HT29) with TPT at a concentration of 10 micromolar for 24 hours resulted in a significantly higher activation of caspase-3/7 when compared to treatment with Cisplatin, indicating a more robust induction of apoptosis by TPT. The enhanced cytotoxicity observed with TPT could potentially be explained by an increased accumulation of active platinum species within the cancer cells. This intracellular accumulation of platinum drugs can be significantly influenced by the overall lipophilicity of the specific platinum compound. Previous studies have indeed described that the introduction of simple ligands into platinum complexes can lead to increased platinum cellular uptake, which is consistently associated with enhanced cytotoxic activity. To investigate this, the lipophilicity index, expressed as the log Pow value, was determined for TPT, yielding a value of -0.41 ± 0.09. This value is consistent with previous data reported for similar compounds and is notably higher than the log Pow of Cisplatin, which was determined to be -2.03 ± 0.47. Given that a higher lipophilicity of TPT could potentially explain its enhanced biological activity, the researchers speculated that a platinum(IV) compound with a comparable log Pow value might exhibit similar biological activity. Consequently, compound 4 was synthesized, as it was reported to possess a log Pow value similar to that of TPT, specifically -0.46 ± 0.04 compared to TPT’s -0.41 ± 0.09. Surprisingly, despite its similar lipophilicity, compound 4 was found to be significantly less cytotoxic than TPT in both A549 lung cancer cells (IC50 = 6.3 micromolar for compound 4 versus 0.5 micromolar for TPT) and HT29 colorectal cancer cells (IC50 = 34.8 micromolar for compound 4 versus 4.7 micromolar for TPT). The reduced activity of compound 4, despite its comparable lipophilicity, suggests that factors beyond simple lipophilicity-enhanced passive transport are at play. This discrepancy could be attributed to decreased platinum(IV) internalization, a slower intracellular reduction rate, or a reduced release of the cytotoxic platinum(II) species from compound 4.

To further unravel the mechanisms underlying TPT’s enhanced activity, the intracellular platinum accumulation of TPT, Cisplatin, and compound 4 was quantitatively investigated using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). A549 lung cancer cells and HT29 colorectal cancer cells were seeded onto multiwell plates and allowed to adhere, then subsequently exposed to Cisplatin, TPT, and compound 4, all at a concentration of 10 micromolar. Platinum uptake was measured after a four-hour treatment period. As hypothesized, cells treated with TPT exhibited a remarkably higher platinum uptake, approximately 11-fold greater than cells treated with Cisplatin. However, cells treated with compound 4 displayed notably poor platinum uptake, despite its similar lipophilicity to TPT. This critical finding strongly suggested that TPT’s enhanced internalization could not be exclusively explained by its increased lipophilicity facilitating passive transport. Consequently, the researchers hypothesized that an active transport mechanism, potentially relying on ATP-powered pumps, might be involved in TPT’s cellular entry. To test this hypothesis, the platinum uptake of TPT and Cisplatin was re-evaluated at a lower temperature of 4 degrees Celsius, a condition known to significantly reduce the activity of ATP-dependent pumps in cancer cells. Interestingly, the ratio of platinum internalization between TPT and Cisplatin dropped considerably at this lower temperature: from an 11-fold difference to approximately a 2-fold difference in A549 cells, and from a 13-fold difference to about a 3-fold difference in HT29 cells. This substantial reduction in the uptake ratio at low temperature strongly supported the hypothesis that TPT could indeed be internalized, at least in part, through an active transport system.

While the precise underlying mechanism of this active transport remains to be fully elucidated, the emerging correlation between signaling originating from the tumor microenvironment—a complex milieu composed of non-malignant cells surrounding the cancer cells—and treatment efficiency, strongly suggests a key role for stromal and immune components in modulating a patient’s response to therapy. Following this crucial rationale, the researchers proceeded to evaluate the impact of TPT and Cisplatin directly on immune cells. Surprisingly, when a macrophage cell line, RAW 264.7, was treated with TPT and Cisplatin, an entirely opposite biological effect was observed compared to cancer cells. Indeed, Cisplatin was found to be significantly more cytotoxic than TPT after both 24 and 72 hours of treatment, indicating TPT’s reduced harmfulness to these immune cells. Intrigued by this inverse cytotoxicity, the researchers next investigated the platinum internalization of TPT and Cisplatin in RAW 264.7 macrophages. Cells were treated with TPT and Cisplatin at a 10 micromolar concentration, and platinum content was determined following a 4-hour treatment. Consistently, cells treated with Cisplatin accumulated significantly higher quantities of platinum than cells treated with TPT. These results collectively reinforce the hypothesis that increased intracellular accumulation of TPT in cancer cells cannot be solely explained by lipophilicity-enhanced passive transport, and instead strongly suggest that other active mechanisms—such as ATP pumps or specific receptors—could be differentially involved in cancer cells versus immune cells. However, further in-depth investigation will be needed to precisely elucidate the complete mechanism of TPT’s internalization and its differential uptake.

Several independent sources have consistently reported a positive influence of macrophages, particularly those located at the tumor invasive front, on the prognosis of patients with colorectal cancer. For instance, studies have shown that increased patient survival is associated with the presence of VEGF-expressing tumor-associated macrophages (TAMs). Conversely, other reports have indicated that a lower number of TAMs correlates with a worse prognosis. Additionally, research has found that patient survival was significantly improved by an increased infiltration of intra-tumoral dendritic cells. More broadly, a comprehensive meta-analysis of 32 studies, collectively encompassing 2988 patients, strongly suggested that a higher tumor inflammatory infiltrate is consistently associated with a better prognosis in patients diagnosed with colorectal cancer. Therefore, the development of therapeutic agents that specifically target colorectal cancer cells while strategically avoiding or minimizing harm to the critical immune infiltrate within the tumor microenvironment holds immense therapeutic interest and potential.

Given that TPT exhibited low cytotoxicity and reduced platinum uptake in the macrophage cell line RAW 264.7, the researchers hypothesized that a similar beneficial biological effect, namely immune cell sparing, could be observed in actual patient tumors using an ex-vivo model. To this end, a sophisticated human tumor explant model was developed. This innovative model meticulously recapitulates, in an *in vitro* setting, the complex intratumoral heterogeneity, encompassing both the cancerous epithelial cells and the surrounding tumor microenvironment, crucially including the inflammatory infiltrate. This unique capability allowed for the direct evaluation of treatments that might differentially affect both cancer epithelial cells and associated stromal/immune cells. In brief, tumor explants obtained directly from surgical specimens of untreated colorectal cancer patients were cultured ex vivo in the presence of TPT (10 micromolar) or Cisplatin (10 micromolar) for a duration of 72 hours. The treatment’s cytotoxic activity was then assessed by the immunohistochemical detection of cleaved caspase-3, which serves as a highly reliable surrogate marker of apoptosis or programmed cell death within the explants.

Intriguingly, immunohistochemical analysis revealed a prominent accumulation of nuclear cleaved caspase-3 reactivity in TPT-treated tumor explants, indicating robust apoptotic activity, particularly when compared to Cisplatin-treated samples. To further analyze the presence and distribution of inflammatory cells within these treatment settings, CD45 immunohistochemistry was performed. CD45 is a widely used pan-leukocyte marker, identifying virtually all immune cells. In the Cisplatin-treated explants, only a few isolated CD45-positive cells were observed in most samples. In contrast, TPT-treated samples showed an overall increased inflammation closely related to the tumor epithelium. More quantitatively, “hotspots” containing significantly higher numbers of CD45-positive cells were identified in TPT-treated explants, specifically demonstrating 57 and 60 CD45-positive cells respectively in high power fields in two out of four explants. Even at a higher chemotherapy concentration (90 micromolar), only one hotspot of 5 CD45-positive cells was detected in explants treated with Cisplatin, whereas explants treated with TPT consistently showed an overall occasional presence of CD45-positive cells and one hotspot of 10 CD45-positive cells. Collectively, these compelling data strongly suggest that Traniplatin induces intense and effective intra-tumoral cytotoxicity against cancer cells, yet this efficacy is remarkably associated with an increased presence of immune cell infiltrate when compared to standard platinum-based chemotherapy like Cisplatin. This pattern of findings points towards a distinctly decreased cytotoxic activity of TPT specifically against immune cells, a highly advantageous characteristic in the complex milieu of the tumor microenvironment.

Conclusions

A novel platinum(IV) pro-drug, systematically named Traniplatin (TPT), has been successfully synthesized and characterized. This innovative compound is strategically based on the well-established chemotherapeutic agent Cisplatin and incorporates the clinically utilized drug Tranilast as an axial ligand. The comprehensive biological evaluation of this newly synthesized TPT demonstrated its enhanced cytotoxic activity against both colorectal and lung cancer cell lines, leading to a more potent induction of programmed cell death as evidenced by the activation of caspase-3/7 and subsequent apoptosis.

Further mechanistic investigations revealed that a significantly higher intracellular uptake of platinum was measured in cancer cells treated with TPT when compared to those treated with Cisplatin. However, this advantageous effect of increased internalization could not be exclusively attributed to TPT’s comparatively increased lipophilicity alone, strongly suggesting the involvement of other active transport mechanisms in its cellular entry within cancer cells. Crucially, a distinctly opposite biological effect was observed in macrophage cell lines, where both the internalization of platinum and the cytotoxicity of TPT were significantly reduced when compared to Cisplatin. This selective sparing of immune cells was further substantiated and mirrored in experiments utilizing human colorectal cancer patient tumor explants treated with TPT, where the compound continued to induce intense intra-tumoral cytotoxicity against cancer cells while simultaneously being associated with an increased presence of intra-tumoral immune cell infiltrate. This compelling observation directly correlates with a reduced cytotoxic activity of TPT against these vital immune cells within a complex biological context.

Previous extensive studies in the field of oncology have consistently reported a robust and positive correlation between the presence of inflammatory cells within the tumor microenvironment and an improved prognosis for patients suffering from cancers of distinct origins, including colorectal cancer. Therefore, a chemotherapeutic agent like TPT, which exhibits decreased cytotoxicity against CD45-positive immune cells while maintaining or enhancing its anti-cancer efficacy, holds profound promise for potentially improving the clinical outcome of treated colorectal cancer patients. The unique dual profile of TPT, characterized by its increased cytotoxicity against cancer cells coupled with a remarkably decreased cytotoxicity against crucial immune cells, positions it as a compound of immense therapeutic interest for future anti-cancer strategies.

Acknowledgments

This research endeavor was made possible through the generous financial support provided by Generalitat de Catalunya, specifically through its support for the XRB network, and by the MINECO-FEDER initiative through the specific grant. Additional critical funding was gratefully received from the Cellex Foundation, the Asociación Española contra el Cáncer (AECC), and the Proyectos de Investigación en Salud (ISCIII). Furthermore, invaluable support was provided by the ‘Xarxa de Bancs de Tumors’, which is sponsored by the Pla Director d’Oncologia de Catalunya. The IRB Barcelona institution itself is a distinguished recipient of a Severo Ochoa Award of Excellence from the MINECO, Government of Spain, recognizing its outstanding research contributions, and it is an integral part of the CERCA network of research centers overseen by the Generalitat de Catalunya. Individual researchers contributing to this work also received specific fellowship support, including a Beatriu de Pinós fellowship for D.L., a Miguel Servet contract from the Instituto de Salud Carlos III for A.C., and a Marie Curie fellowship for M.D. The authors express their sincere gratitude for the excellent and dedicated assistance provided by the Histopathology core facility at IRB Barcelona, and specifically thank Iris Joval for her invaluable assistance in the preparation and mounting of the figures presented in this study.