The in vitro immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity is increased by hypofractionated irradiation

With approximately 70.000 new cases of disease per year, breast cancer (mamma carcinoma) represents the most frequent and, along with approximately 17.000 deaths per year, also the deadliest cancer disease for women in Germany. One out of 8 German women will suffer from mamma carcinoma during lifetime. This implies that deep knowledge about breast cancer development, mechanisms of tumor progression and related treatments is mandatory. The main risk factors to develop a mamma carcinoma are female gender and seniority (>60 years). Breast cancer displays a heterogeneous tumor disease and multiple subtypes exist [1]. Ductal, originating from lactiferous ducts, are to be differed from lobular carcinomas, originating from glandular lobes. With about 70 % of the cases the invasive ductal carcinoma is the prominent type [2]. Precancerous conditions are the Ductal Carcinoma in situ (DCIS) and the Carcinoma Lobulare in situ (CLIS), of which the DCIS shows the more aggressive progress and in about a third to half of the cases develops to an invasive carcinoma within 10–20 years [3]. Benign and malignant pre-existing conditions of the breast, genetic mutations, most prominent in the BRCA (Breast Cancer) gene, positive family history, long period of estrogen-exposure (early menarche, late menopause, obesity) and life style are main risk factors [4].

Triple negative breast cancer (TNBC) represents 15–20 % of all breast cancers that lack estrogen receptor (ER) and progesterone receptor (PgR) expression as well as amplification of the human epidermal growth factor receptor 2 (HER2). TNBCs are an aggressive group of breast cancers with higher rates of relapse and to date not a single targeted therapy has been approved for its treatment [5]. Combinational effects of chemotherapy, photothermal therapy, and gene therapy with low drug dose are currently tested as promising strategy for TNBC treatment [6]. However, a relative radioresistance for TNBC does not imply radiation omission, because radiotherapy (RT) provides an absolute loco-regional risk reduction [7].

RT is therefore a crucial component for the treatment of breast cancer [8]. Commonly it is applied in daily fractions of 1.8–2 Gy up to a total dose of 50 Gy [9]. However, long term follow-up confirms that appropriately dosed hypofractionated radiotherapy is safe and effective for patients with early breast cancer [10]. Meanwhile, the use of fractions >2.0 Gy (hypofractionation) is standard in the UK, and increasingly used internationally for this tumor entity [11]. The results of the German multicenter phase II trial (ARO-2010-01) also suggest that hypofractionation with simultaneous integrated boost for early breast cancer is feasible [12]. However, integration of RT in multimodal breast cancer treatment still remains a challenge [13].

Emerging evidence suggests that besides inducing local DNA damage, RT promotes a pro-immunogenic milieu within the tumor capable of stimulating host cancer-specific immune responses. Immunogenic breast cancer cell death stimulated by either high-dose RT alone or concurrent chemoradiation regimens may contribute to this [14]. Especially combination of RT with further immune stimulation contributes to immune-mediated abscopal effects of RT [15, 16]. Recent in vitro and in vivo studies indicate a unique immune modulating prospect of hyperthermia (HT), especially when combined with RT [17].

Regional or local HT describes the overheating of certain body parts by using electromagnetic radiation, e.g. microwaves. Temperatures of 40–44 °C for a period of at least 60 min should be achieved for therapeutic effectiveness [18]. Of note is that recommendations for the implementation of quality-assured hyperthermia treatments should be followed when applying HT in multimodal therapy settings [19]. A radiosensitizing effect of HT for the treatment of breast cancer has been discussed for long time [20, 21]. In the update of 2012 of the AGO German interdisciplinary S3-guidelines for diagnostic, therapy and follow-up care of breast cancer it is stated that for chest wall recurrences, simultaneous chemotherapy or hyperthermia as radiosensitizing methods may result in higher response rates. For re-irradiation, combination of RT with HT received the Oxford Levels of Evidence of 1b in these guidelines. A Meta-analysis of 23 studies with 1861 patients with tumors of the chest wall, cervix, rectum, bladder, prostate, head-neck-region and melanoma points out a highly significant benefit regarding loco-regional tumor control when RT is combined with HT [22].

Oldenborg and colleagues proposed and examined a hypofractionated irradiation with a single dose of 4 Gy or 3 Gy, respectively, twice a week plus HT for patients with recurrent breast cancer in previously irradiated area [23]. Of note is that no increasing toxicity was observed when adding HT to RT for the treatment of high risk breast carcinoma [24]. Re-irradiation in combination with HT is an effective and a safe modality to treat loco-regional recurrences of breast cancers [25]. Besides HT, also electrochemotherapy is discussed to be beneficial for chest wall breast cancer recurrence [26].

The dogma of classical radiobiology states that the cytotoxic effects of irradiation on tumor cells are due to induction of DNA double strand breaks. The colony forming assay is used as gold standard to determine the “viability” of cells as defined by bearing the capability to form colonies >50 cells. However, no predictions about cell death of a single cell and forms of tumor cell death including their immunogenicity can be drawn by this assay [27], besides several other draw backs such as seeding the cells from the beginning in different concentrations in dependence of the treatment, disregarded cell-to-cell communication, clump artifacts and unification of colonies of different sizes [28].

None repaired DNA damage can lead to cell death via apoptosis, mitotic catastrophe, autophagy, or terminal growth arrest senescence [29]. Defining life and death is more problematic than one would guess and regulated cell death often results in initiation of secondary regulated cell death accompanied by inflammatory events [30]. The success of RT does therefore not only derive from direct cytotoxic effects on the tumor cells alone, but instead might also depend on resulting innate as well as adaptive immune responses [31]. The latter can be initiated by immunogenic cell death forms such as necrosis (summarised in [32]).

While apoptotic cells when present in physiological amounts exert non- or even anti-inflammatory effects [33], primary and secondary necrotic cells lead to an activation of the immune response by release of danger signals such as high mobility group box protein 1 (HMGB1), Adenosine-Triphosphate (ATP) and/or heat shock protein 70 (Hsp70) (summarised in [34]). Secondary necrotic cells derive from apoptotic ones that have lost their membrane integrity during the course of cell death execution [35]. Therefore they are also often termed as late apoptotic cells. However, following the definition of necrosis, namely the loss of membrane integrity, secondary necrosis is the better fitting term from the immunological point of view.

Central in immune activation against the tumor by therapy-induced immunogenic tumor cell death forms are dendritic cells (DCs). High numbers of apoptotic cells and danger signals released by primary and secondary necrotic cells trigger maturation and activation of DCs [36, 37]. The latter then present tumor-derived antigen to CD8+ T cells and thereby initiate cytotoxic T cell responses against the tumor (summarised in [38]).

Hence, based on the clinical launching of hypofractionated RT and addition of HT as radiosensitizer for the treatment of breast cancer, our study opened the question whether cell death forms and immunogenic potential of breast cancer cells differs after hypofractionated RT and/or HT. We focused on the MDA-MB231 breast cancer cell line representing triple-negative mesenchymal highly invasive human breast cancer cells and MCF-7 breast cancer cells, being positive for ER and PgR expression, but deficient for caspase-3, for comparison. As activation of caspases is widely considered as the initiator mechanism of regulated cell death [30], we especially focused on the role caspase-3 in induction of cell death with immunogenic potential of breast cancer cells.

Although the last decades brought a wide enlargement of breast cancer therapies, there are still several limitations which need to be vanquished [50]. Advances in chemo- and radiotherapy, surgical techniques and the development of new targeted agents have significantly improved clinical outcomes for patients with locally advanced breast cancer [51]. Radiotherapy is an integral part of the therapy for local tumor control and we observed that the triple negative but caspase 3 proficient MDA-MB231 cells are less radiosensitive than the MCF7 cells regarding the clonogenicity (Fig. 1). However, higher amounts of apoptotic and necrotic cells are induced by irradiation starting at 4Gy in MDA-MB231 cells compared to MCF-7 cells (Fig. 2). This highlights that definition of “radiosensitivity” is diverse in dependence of the focus on tumor cells clonogenicity or cell death, respectively, and that cell death is dispensable for radiation-induced reduction of clonogenicity [27, 52]. Along the same line, while HT did not impact on cell death induction (Fig. 3), it slightly, but significantly reduced the surviving fraction based on the clonogenicity of MDA-MB231 cells (Fig. 1c). Further, addition of HT to hypofractionated RT reduced the amount of cells in the radiosensitive G2 cell cycle phase only in MDA-MB231 slightly, but significantly (Additional file 1: Figures S1). This could indicate that p53 mutated tumor cells such as MDA-MB231 response to HT by reduction of radiation-induced G2 arrest, as also observed for checkpoint kinase inhibitors [53].

While for the aggressive triple-negative breast cancer subtype innovative treatment methods based mainly on chemotherapy, biologic agents and radiotherapy show also good tumor control, a limited time of disease free survival is still the reality [54]. This implies that especially systemic immune activation might be beneficial for fighting metastases and recurrent tumors [38]. For activation of the immune system the endoplasmatic reticulum (ER) stress and forms of tumor cell death are decisive and not the clonogenicity of the tumor cells themselves [55, 56]. Hypofractionated irradiation was used in our preclinical in vitro assays, since 10 years follow-up confirmed that hypofractionated RT is safe and effective for patients with early breast cancer [10]. In preclinical model systems, abscopal and immune mediated effects in breast cancer are mainly observed when combining hypofractionated irradiation with further immune stimulation [57]. We focused on hyperthermia as a promising enhancer of RT concerning direct tumor cell cytotoxicity, but also as a stimulator of the immune system by generating an in situ tumor vaccine [17]. Here, DCs are central since they link innate and adaptive immune responses and are stimulators of CD8+ cytotoxic T cell responses. Such tumor infiltrating lymphocytes (TILs) have been demonstrated to predict higher pathologic complete response rates in triple negative patients [58, 59].

Our data suggest that caspase-3 proficient MDA-MB231 can be rendered more immunogenic by hypofractionated irradiation. Treatment of these cells with ionizing radiation resulted in a mixture of apoptotic and necrotic cells at doses of 4Gy and above (Fig. 3a and b). This mixture of dying and dead tumor cells is supposed to bear a high immunogenic potential [60]. When MDA-MB231 cells were treated with 4x4Gy or 6x3Gy hypofractionated irradiation, also a mixture of apoptotic and necrotic cells resulted (Figs. 4 and 6). HT did impact on the percentage of cells with subG1 content (Fig. 4d) and the release of the danger signal Hsp70 (Figs. 5b and 7b): both were increased when HT was added to hypofractionated RT in MDA-MB231 cells.

In MCF-7 cells, cell death induction by RT was much weaker and HT did not increase the amount of late apoptotic cells with increased subG1 DNA content nor resulted in release of Hsp70 or HMGB1. This could suggest that especially combination of hypofractionated RT with HT in caspase-3 proficient cells such as MDA-MB231 cells generates tumor cell SNs that lead to increased expression of activation markers on DCs. However, the expression of the CD80 and CD86 on DCs was increased when incubating immature DCs with SNs of MDA-MB231 cells that had been exposed to hypofractionated irradiation. Addition of HT did not significantly impact on it (Fig. 8b). Concomitantly, a decreased expression of DC-SIGN was detected. Since ligation of DC-SIGN on DCs actively primes DCs to induce Tregs, a reduced expression is favorable for induction of anti-tumor immune responses [61]. Regarding the homing receptor CCR7 on DCs, also a slight, but not significant, increased expression was observed after hypofractionated RT (not shown). This is favorable for directing the DCs to the next lymph nodes where they then prime naïve T cells [62]. We have previously shown that the SNs of colorectal tumor cells that had been treated with RT plus HT also induce a significant up-regulation of expression of CD80 and CCR7 on DCs and that Hsp70 is one mediator of it. However, Hsp70 is not the sole stimulus for this, since HT treatment alone resulted in a comparable release of Hsp70 compared to RT plus HT, while only a very slight increase of expression of CD 80 and CCR-7 on DCs was observed in the case of only HT application [44]. In the case of the MDA-MB231 breast cancer cells, slightly higher concentrations of Hsp70 in tumor cell SNs after combination of fractionated RT with HT is not the key stimulus for increased surface expression of activation markers on DCs (Fig. 8). This necessitates that further immune stimulatory molecules besides Hsp70s, which have to be identified in future work, are released by the breast cancer cells. HMGB1 might be one component of it, since its concentration is slightly increased in MDA-B231 cells, but not in MCF-7 cells, only in dependence of irradiation (Fig. 7a). The increased amount of Hsp70 after combination of hypofractionated RT with HT might rather lead to activation of NK cells than to further stimulate DCs [63, 64].

Caspases are central for most apoptosis inductions [65]. We proved that apoptosis and consecutive secondary necrosis-induction by hypofractionated irradiation of breast cancer cells is dependent on caspase-3 and can therefore be efficiently blocked by the pan-caspase inhibitor zVAD-fmk (Fig. 6). We conclude that hypofractionated RT induces cell death only in caspase-3-proficient breast cancer cells. Addition of HT to hypofractionated RT fosters the release of Hsp70 by these cells, but does not generate tumor cell SNs that do further increase the hypofractionated radiation-dependent enhanced expression of activation markers on DCs (Fig. 8).

Nevertheless, the multiple in vitro assays that we used for our study allow to draw the conclusion that hypofractionated irradiation is one main stimulus for cell death induction and consecutive DC activation in caspase-3 proficient breast cancer cells. Hennel et al. demonstrated that ablative RT, also as single application, potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53 [66]. This in turn stimulates monocyte migration and most likely consecutive immune activation. Therefore, both, radiation with higher single doses applied in ablative and hypofractionated RT seem to be a key stimulus for DC activation by SNs of caspase proficient tumor cells.

Additional immune modulation with HT fosters the release of higher amounts of Hsp70 and therefore might result in activation of NK cells. Since synergistic effects of innate and adaptive immunity are beneficial for the induction of anti-tumor immunity and especially for immunological memory [67] it should also be the aim to activate both CD8+ T cells via DCs and NK cells for therapy of selected breast cancer types. Besides consideration of disease extent, host factors, patient preferences and social and economic constraints [68], the configuration of tumor cells with cell death executing proteins such as caspases should be included in radio-immunotherapy decisions to enhance the immunogenicity of distinct breast cancers by hypofractionated RT alone or in combination with further immune modulation in a personalized manner.