Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model

Breast cancer remains one of the four major cancers, and is expected to account for approximately 30% of all new cancer diagnoses in women in the USA, making it the leading cause of cancer-related deaths in women aged 20–59 years [1]. Triple-negative breast cancer (TNBC) represents 15% of breast carcinomas, a particularly aggressive and heterogeneous subtype defined by the absence of the three primary breast cancer biomarkers - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2, also known as ERBB2). Triple-negative breast cancer is recognized by high proliferative activity, increased immune cell infiltration, and a more basal-like and mesenchymal phenotype. Patients with TNBC do not benefit from endocrine or anti-HER2 therapy, leaving chemotherapy as the only established option [2]. Resistance usually develops after an initial period of response [3]. Hence, there is an urgent need for innovative treatment options that can improve disease outcome or complement existing therapies for patients with breast cancer. With the introduction of immune-based therapies, such as checkpoint blockade, significant advances have been made in the treatment of cancer, thereby illustrating the importance of harnessing the immune system. However, immunotherapy has shown limited success in the treatment of breast cancer [4]. The next step to further increase the clinical benefit of breast cancer therapy will be to target the antitumor immune response at multiple levels, which may be accomplished through combination therapy approaches. Turning cold tumors hot would further augment the antitumor efficacy of established therapies such as checkpoint blockade [5]. By using immune-based combination therapy it is possible to shift the clinical response to a durable response due to immunological memory [6].

Host defense peptides, commonly denoted as cationic antimicrobial peptides (CAPs) [7], often possess anticancer and immune-modulating properties due to their amphipathic nature and cationic charge. Several CAPs have been shown to target cancer cells by interacting with and destabilizing anionic lipid membranes or highly negatively charged intracellular targets such as mitochondria [8], thus emphasizing their potential as novel anticancer agents [9‐15]. We have previously described the development of LTX-315, a first-in-class oncolytic peptide used for intratumoral treatment of solid tumors [16, 17]. The mode of action is associated with a reshaping of the tumor microenvironment, in which LTX-315 induces immunogenic cell death through a membranolytic effect (necrosis), as well as direct effects on intracellular targets such as mitochondria [18‐21]. Changes to the tumor microenvironment are dependent on the release of potent danger-associated molecular pattern molecules (DAMPs) and tumor antigens that recruit and activate dendritic cells with subsequent T-cell activation and tumor-specific immune responses. Intratumoral treatment with LTX-315 results in growth inhibition, complete regression and long-lasting tumor-specific immune responses in a variety of different experimental animal models [22‐24].

Conventionally, chemotherapeutic agents have been used in the clinic based on their capacity to directly kill proliferating cells. However, although high-dose chemotherapy has been shown to significantly debulk the tumor, it often leads to disease relapse and the establishment of drug resistance. There is now a growing focus on a new therapeutic paradigm using low-dose or medium-dose chemotherapeutics at short repeated intervals due to their potential to stimulate anticancer immune responses while selectively eliminating immunosuppressive cells [25]. For this reason, chemotherapy may be a promising candidate in combination with immunotherapy, defined as immunochemotherapy, as shown by augmented anticancer effects following treatment with chemotherapy and anticancer vaccines [26, 27]. Furthermore, doxorubicin has been shown to induce immunogenic cell death in addition to inhibit suppressive cells such as myeloid-derived suppressor cells, thereby removing the brakes implemented on the immune system by such cells [28‐30].

In the present study, we investigated the potential of combining intratumoral treatment with LTX-315 and CAELYX® (liposomal doxorubicin) in a preclinical TNBC model. The unique mode of action of LTX-315, reprogramming and reshaping the tumor microenvironment, thus turning “cold” tumors into “hot” tumors, showed a significant additive antitumor effect when combined with CAELYX®. It is well-established that tumor-infiltrating CD8⁺ T cells are important for therapeutic efficacy, and help predict clinical outcome in breast cancer [5, 31]. LTX-315 induced an increase in the infiltration of cytotoxic CD8⁺ T cells into the viable tumor bed, creating an immunogenic antitumor microenvironment. Combining LTX-315 with doxorubicin further augmented this effect.

Cancer therapy has been transformed with recent advances in immunotherapy. Nonetheless, further therapeutic advances are needed, with efforts focusing on implementing strategies that combine different cancer treatments. Combination therapy has the potential to dramatically improve the outcome in patients with cancer by harnessing potential synergies that can lead to better clinical responses and increased survival. Over time it is expected that combination strategies will become the standard of care for all cancers, although the discovery of effective combinations is a challenging endeavor [32]. One promising strategy is the combination of immunotherapy and chemotherapy, using immunotherapeutic drugs capable of activating antitumor immunity in combination with chemotherapeutic drugs capable of inducing immunogenic cell death (ICD), while at the same time inhibiting cancer-mediated immunosuppression [29].

Triple-negative breast cancer (TNBC) is recognized as a heterogenous, biologically aggressive disease for which there are few treatment alternatives. There are several promising therapeutic strategies being explored in phase I–III clinical studies, but conventional chemotherapy is still considered the standard of care. Patients with chemo-resistant TNBC have a poor prognosis, and often a very rapid onset of metastasis. However, the highly proliferative status of TNBC also makes it more sensitive to chemotherapy, hence making it a good candidate for combination strategies involving chemotherapy and immunotherapy, since tumors often have an increased immune cell infiltrate [2, 4].

LTX-315 is a de novo designed oncolytic peptide for local treatment of solid tumors, capable of reshaping and reprogramming the tumor microenvironment by ICD [16, 33]. The peptide has a dual mode of action, inducing cancer cell lysis, by destabilizing and disintegrating the cellular membrane, and interacting with intracellular targets such as the mitochondria [18, 20].

To further study the potential of LTX-315 in breast cancer therapy, orthotopical 4 T1 mammary carcinomas were established in syngenic Balb/C mice, and treated with LTX-315 and CAELYX® according to the experimental setup illustrated in Fig. 1. In animals, the 4 T1 cells had highly aggressive properties, forming rapidly growing carcinomas metastasizing to the lungs. All control animals were euthanized before day 30 (Fig. 2b) due to tumor burden or lung metastasis. Sub-therapeutic treatment with LTX-315 did not inhibit tumor growth significantly, whereas CAELYX® alone partially inhibited tumor growth, and resulted in a slight increase in overall survival (Fig. 2b). Thus, the therapeutic efficacy of either monotherapy was limited. Interestingly, when LTX-315 and CAELYX® were used in combination, tumor growth was significantly inhibited and complete tumor regression was achieved in 50% of the animals, indicating a strong additive antitumor effect between LTX-315 and CAELYX® (Fig. 2b), which was verified by statistical analysis of the different treatment groups (p ≤ 0.05). In the combination group, 50% of the treated animals were tumor free, as demonstrated by BLI (data not shown) at the end of the study on day 80 (Fig. 2c). Altogether, this suggests a strong and long-lasting antitumor response able to eradicate both local tumor growth and metastasis.

The tumor debulking property of LTX-315 was quantified using BLI and MRI (Fig. 3a and b, respectively). The MRI demonstrated large necrotic areas within the tumor microenvironment on day 14 and 21, both with LTX-315 alone and in the combination group (Fig. 3b). Comparably, CAELYX® alone inhibited tumor growth temporarily, indicating a partial response before recurrence on day 28. CAELYX® alone did not induce significant tumor debulking. LTX-315 in combination with CAELYX® induced complete regression of 4 T1 mammary fat pad carcinomas in several of the treated animals (Fig. 3a and b). Images from BLI exhibited significant differences in signal intensity related to the treatment given, thus a scale bar was not applicable. Treatment with LTX-315 induced local necrosis, leading to leakage and loss of BLI signal, even though tumors recurred later. For this reason, the intensity of the BLI signal did not always reflect the measured tumor size and BLI was used primarily as a method for monitoring metastasis.

To elucidate the mechanism behind the complete regression of tumors treated with the combination, tumor tissue was analyzed by immunohistochemistry (IHC) and flow cytometry. Tumor samples for IHC (n = 4) demonstrated that combination therapy with LTX-315 and CAELYX® induced significant necrosis and hemorrhagic damage in the 4 T1 TNBC tumors. In addition, the combination therapy resulted in an increase in tumor-infiltrating CD8+ T cells into the remaining viable tumor tissue on day 6 post treatment. On day 27, a majority of the combination animals were tumor free as illustrated by histological samples showing no remaining viable tumor tissue (Fig. 4).

Flow cytometry (n = 8) showed that the combination treatment induced extensive tumor cell killing with very few live cells 7 days post treatment (Fig. 5a). Furthermore, treatment with LTX-315 alone resulted in a significant increase in CD45+ immune cells into the tumor parenchyma. CD45 is expressed on all leukocytes, including neutrophils and natural killer (NK) cells [34]. Given that LTX-315-treatment results in a substantial increase in CD45+ cells compared to the other treatment groups (Fig. 5b), we speculate that immune cells other than CD8+ T cells are also important in the LTX-315-induced antitumor immune response. These may include CD4+ T helper cells and NK cells. Natural killer cells have been shown to have an important role in cancer immunotherapy and are capable of interacting with and killing cancer cells directly by numerous modes of action [35]. Treatment with CAELYX® alone reduced the amount of CD4+ T cells in the tumor microenvironment on day 13 and 14, as demonstrated by both IHC and flow cytometry (Figs. 4 and 5d, respectively). Thus, the additive antitumor effects observed when LTX-315 is combined with CAELYX® may be partially explained by a reduction in immunosuppressive cells, such as CD4+ regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Even though little is known about the effect of doxorubicin on Tregs, doxorubicin has been shown to eliminate myeloid-derived suppressor cells (MDSCs) [28]. Moreover, the down-regulation of CD4+ T cells following treatment with CAELYX® alone seems to be a transient process, as demonstrated by an increase in CD4+ T cells on day 27 post treatment (Fig. 4). Treatment with LTX-315 alone and the combination induced an increase in tumor-infiltrating CD4+ T cells on day 13 and 14 (Figs. 4 and 5d). The increase in tumor-infiltrating CD4+ T cells following treatment with LTX-315 alone and the combination, may indicate reshaping of the tumor microenvironment from immunosuppressive CD4+ Tregs to a different CD4+ T cell phenotype important for antitumor immunity. CD4+ T helper cells have indeed been shown to be a critical element in optimal activation of CD8+ T cells and in the maintenance of cancer-related immune memory [36‐38].

CAELYX® alone induced significant infiltration of CD8+ cytotoxic T cells compared to the other treatments (Fig. 5e). The increase in tumor-infiltrating CD8+ T cells may be related to the amount of viable tumor tissue left in the treated tumors 7 days post treatment. In the LTX-315 alone and in the combination group most of the tumor tissue was necrotic following treatment, compared to tumors treated with CAELYX® alone, which were less necrotic. In this study, and in previous studies, we have observed that tumor tissue with substantial necrosis following intratumoral treatment does not contain significant numbers of CD8+ T cells in the necrotic region of the tumor, when analyzed by IHC. Accordingly, we observe extensive infiltration of CD8+ T cells residing in the remaining viable tumor tissue. Hence, it is thought that massive necrosis and tumor debulking will lead to a smaller total number of active CD8+ T cells inside the tumor tissue area when analyzed in a quantitative manner.

It is well-established that the location of immune cells is equally important to the total number of immune cells, when it comes to the assessment of immunoscore and patient prognosis and survival [39]. In our study we observed differences in tumor-infiltrating CD4+ and CD8+ T cells following treatment with the combination, when analyzed by IHC and flow (Figs. 4 and 5). Considering IHC is a snapshot of a specific region of the tumor parenchyma, while flow cytometry investigates the total tissue harvested, this is not unexpected. Analysis with IHC was focused on areas of interest, such as the region between the treatment-induced necrosis and the viable tumor tissue. This is the area where CD4+ and CD8+ T cells will exert their cytotoxic antitumor activity as they gain access to viable tumor cells. Contrarily, flow cytometry examines the total tissue harvested and does not differentiate between tumor-excluded or tumor-infiltrating CD4+ and CD8+ T cells. While IHC is a method that evaluates compartmentalization of immune cells, which is very important for immunoscore and clinical response, flow cytometry does not. Thus, it is important to analyze the potential reshaping of the tumor microenvironment in both a qualitative (IHC) and a quantitative (flow cytometry) manner.

Altogether, these data indicate strong reshaping of the tumor microenvironment in treated lesions versus non-treated lesions, thereby resulting in an immune-mediated antitumor response. This reshaping of the tumor microenvironment is dependent on the immunomodulating properties of both LTX-315 and doxorubicin. Conventional chemotherapeutic agents, including doxorubicin, can stimulate the immune system by directly activating CD8+ or γδ T cells, leading to an antitumor T_H1 response through production of interferon gamma (IFNγ) and IL-17 (reviewed in [40]).

LTX-315 promotes strong local necrosis and ICD through the release of DAMPs, such as ATP, high mobility group box 1 protein (HMGB1) and cytochrome c. DAMPS are important for immune cell activation, antigen presentation, and T cell clonal expansion [16, 18, 40, 41]. This will further induce an intratumoral T cell response shown by an increase in CD3+ tumor-infiltrating lymphocytes (TILs) into the tumor parenchyma [22, 23]. Simultaneously, CAELYX® (doxorubicin) is known to further promote ICD [42] and inhibit the frequency of immunosuppressive cells, such as MDSCs [28]. Furthermore, low-dose metronomic doxorubicin has been shown to inhibit angiogenesis, and inhibit the recruitment of tumor-associated macrophages and cells with tumor-initiating and treatment-resistant properties [43]. Thus, the additive effect between LTX-315 and CAELYX® may arise from the ability of LTX-315 to reshape “cold” tumors into “hot” tumors by stimulating immune activation and an increase in TILs, which is further augmented by doxorubicin’s ability to inhibit angiogenesis, induce T cell infiltration, and deplete immunosuppressive cells [43]. This is also supported by recent evidence demonstrating the importance of TILs in response to therapy and improvement in prognosis in patients with breast cancer [5, 31, 44, 45]. Therefore, the combination of LTX-315 and CAELYX® demonstrates a potential to alter the tumor microenvironment in TNBC by converting it from an immunologically suppressive microenvironment to a more immunologically antitumorigenic microenvironment.

To elucidate the potential of LTX-315 in combination with CAELYX® as a neoadjuvant treatment, 4 T1 breast carcinomas were treated 6 days before tumors were surgically excised from the animals (Fig. 1, experiment 2). At this point, several of the animals already had metastasis, as demonstrated by BLI (data not shown). Most of the animals in this study were euthanized due to metastasis (to the lungs), indicating that a significant increase in survival occurred due to inhibition of metastasis. Our data demonstrate that LTX-315, in combination with CAELYX® and surgery, induced a 60–97% increase in median survival compared to controls or either of the monotherapies and surgery (Fig. 6).

The mode of action of LTX-315 leads to the release of potent immune stimulators (DAMPs) and tumor antigens, which results in tumor-specific antitumor immune responses. For this reason, LTX-315 is an ideal candidate for combination with therapies capable of removing tumor-induced immune suppression implemented by suppressive cells such as MDSCs and Tregs. The current study verifies that LTX-315 has promising potential to be used in immunochemotherapy, and that such combinations warrant further investigation, aiming for future clinical implementation.