Impacts of combining anti-PD-L1 immunotherapy and radiotherapy on the tumour immune microenvironment in a murine prostate cancer model

Background Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. Methods Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. Results 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. Conclusion 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.

FKBPL-based peptide, ALM201, targets angiogenesis and cancer stem cells in ovarian cancer

Background ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. Methods In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). Results ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. Conclusion FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

In vitro and in vivo evaluation of electrochemotherapy with trans-platinum analogue trans-[PtCl2(3-Hmpy)2]

BackgroundCisplatin is used in cancer therapy, but its side effects and acquired resistance to cisplatin have led to the synthesis and evaluation of new platinum compounds. Recently, the synthesized platinum compoundtrans-[PtCl2(3-Hmpy)2] (3-Hmpy = 3-hydroxymethylpyridine) (compound2) showed a considerable cytotoxic and antitumour effectiveness. To improve compound2cytotoxicityin vitroand antitumour effectivenessin vivo, electroporation was used as drug delivery approach to increase membrane permeability (electrochemotherapy).Materials and methodsIn vitro, survival of sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive) was determined using a clonogenic assay after treatment with compound2or cisplatin electrochemotherapy.In vivo, the antitumour effectiveness of electrochemotherapy with compound2or cisplatin was evaluated using a tumour growth delay assay. In addition, platinum in the serum, tumours and platinum bound to the DNA in the cells were performed using inductively coupled plasma mass spectrometry.ResultsIn vitro, cell survival after treatment with compound2electrochemotherapy was significantly decreased in all tested sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive). However, this effect was less pronounced compared to cisplatin. Interestingly, the enhancement factor (5-fold) of compound2cytotoxicity was equal in cisplatin-sensitive TBLCl2 and cisplatin-resistant TBLCl2Pt cells.In vivo, the growth delay of subcutaneous tumours after treatment with compound2electrochemotherapy was lower compared to cisplatin. The highest antitumour effectiveness after cisplatin or compound2electrochemotherapy was obtained in TBLCl2 tumours, resulting in 67% and 11% of tumour cures, respectively. Compound2induced significantly smaller loss of animal body weight compared to cisplatin. Furthermore, platinum amounts in tumours after compound2or cisplatin electrochemotherapy were approximately 2-fold higher compared to the drug treatment only, and the same increase of platinum bound to DNA was observed.ConclusionsThe obtained resultsin vitroandin vivosuggest compound2as a potential antitumour agent in electrochemotherapy.

Adjuvant TNF-α therapy to electrochemotherapy with intravenous cisplatin in murine sarcoma exerts synergistic antitumor effectiveness

Background. Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma.Materials and methods. In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemotherapy with different cisplatin doses.Results. A synergistic interaction between TNF-α and electrochemotherapy was observed. Administration of TNF-α before the electrochemotherapy resulted in longer tumour growth delay and increased tumour curability, and was significantly more effective than TNF-α administration after the electrochemotherapy. Tumour analysis revealed increased platinum content in tumours, TNF-α induced blood vessel damage and increased tumour necrosis after combination of TNF-α and electrochemotherapy, indicating an anti-vascular action of TNF-α. In addition, immunomodulatory effect might have contributed to curability rate of the tumours.Conclusion. Adjuvant intratumoural TNF-α therapy synergistically contributes to electrochemotherapy with intravenous cisplatin administration. Due to its potentiation at all doses of cisplatin, the combined treatment is predicted to be effective also in tumours, where the drug concentration is suboptimal or in bigger tumours, where electrochemotherapy with intravenous cisplatin is not expected to be sufficiently effective.

In vitro and in vivo studies of new cationic Zn(II)-benzonaphthoporphyrazines as photosensitizers for PDT

Four recently synthesized cationic zinc(II)-benzonaphthaporphyrazines 1–4 were studied in vitro and in vivo for their photodynamic therapy (PDT) effectivity. The photophysical measurements showed that in solution and in Cremophor micelles all examined compounds exhibit very high absorption intensity in the spectral range between 680 to 750 nm. The fluorescence emission for 3 and 4 was very well expressed in different media, as well as in cell culture. The dark toxicity examinations on invasive human bladder carcinoma cell line EJ did not show any traces of toxicity. The investigations connected with the detection of their phototoxic capacity on the same cell line demonstrated very promising results especially with photosensitizers 3 and 4. The in vivo studies with these two compounds demonstrating high cell-phototoxic effect were carried out against Lewis lung carcinoma in mice after incorporation in Cremophor micelles. The excitation was done at the respective maximum absorption wavelength for each of the sensitizers at a fluence rate of 380 mW cm-2 and a fluence of 360 J cm-2. The phototherapeutic effect was evaluated through macroscopic observations (tumour growth delay) and by electron microscopy detection. According to these approaches the best effect (including tumour destroyment) was detected after PDT treatment with the cationic tribenzonaphthoporphyrazinato-zinc(II) 3. Typical features of random, but not of programmed, cell death necrosis were observed.