T‐cell acute lymphoblastic leukemia (T‐ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin‐1‐derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first‐described serum‐stable CD47‐agonist peptide) on CEM and MOLT‐4 human cell lines (T‐ALL) and on one T‐murine tumor lymphoblast cell‐line (L5178Y‐R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non‐tumor cells after CD47 activation. In vivo, we determined that PKHB1‐treatment in mice bearing the L5178Y‐R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage‐associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high‐mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase‐independent and calcium‐dependent cell death in leukemic cells while sparing non‐tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Background Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells. Methods Immunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice. Results ICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes. Conclusions ICRP induces ICD in BC cells, leading to long-term antitumour memory.

Acute lymphocytic leukemia (ALL) is the most common pediatric cancer. Currently, treatment options for patients with relapsed and refractory ALL mostly rely on immunotherapies. However, hematological cancers are commonly associated with a low immunogenicity and immune tolerance, which may contribute to leukemia relapse and the difficulties associated with the development of effective immunotherapies against this disease. We recently demonstrated that PKHB1, a TSP1-derived CD47 agonist peptide, induces immunogenic cell death (ICD) in T cell ALL (T-ALL). Cell death induced by PKHB1 on T-ALL cell lines and their homologous murine, L5178Y-R (T-murine tumor lymphoblast cell line), induced damage-associated molecular patterns (DAMPs) exposure and release. Additionally, a prophylactic vaccination with PKHB1-treated L5178Y-R cells prevented tumor establishment in vivo in all the cases. Due to the immunogenic potential of PKHB1-treated cells, in this study we assessed their ability to induce antitumor immune responses ex vivo and in vivo in an established tumor. We first confirmed the selectivity of cell death induced by PKBH1 in tumor L5178Y-R cells and observed that calreticulin exposure increased when cell death increased. Then, we found that the tumor cell lysate (TCL) obtained from PKHB1-treated L5178YR tumor cells (PKHB1-TCL) was able to induce, ex vivo, dendritic cells maturation, cytokine production, and T cell antitumor responses. Finally, our results show that in vivo, PKHB1-TCL treatment induces tumor regression in syngeneic mice transplanted with L5178Y-R cells, increasing their overall survival and protecting them from further tumor establishment after tumor rechallenge. Altogether our results highlight the immunogenicity of the cell death induced by PKHB1 activation of CD47 as a potential therapeutic tool to overcome the low immunogenicity and immune tolerance in T-ALL.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunostimulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by T cells and is associated with improved prognosis in patients affected by triple negative breast cancer (TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces mitochondrial alterations, ROS production, intracellular Ca 2+ accumulation, as well calcium-dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1 (PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover, PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression; both applications induced a long-term antitumor response. Altogether our data shows that PKHB1, a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation through immunogenic cell death induction in breast cancer cells.