Aluminium Nanoparticles as Efficient Adjuvants Compared to Their Microparticle Counterparts: Current Progress and Perspectives.

Aluminium (Al) compounds are used as adjuvants in human and veterinary prophylactic vaccines due to their improved tolerability compared to other adjuvants. These Al-based adjuvants form microparticles (MPs) of heterogeneous sizes ranging from ~0.5 to 10 µm and generally induce type 2 (Th2)-biased immune responses. However, recent literature indicates that moving from micron dimension particles toward the nanoscale can modify the adjuvanticity of Al towards type 1 (Th1) responses, which can potentially be exploited for the development of vaccines for which Th1 immunity is crucial. Specifically, in the context of cancer treatments, Al nanoparticles (Al-NPs) can induce a more balanced (Th1/Th2), robust, and durable immune response associated with an increased number of cytotoxic T cells compared to Al-MPs, which are more favourable for stimulating an oncolytic response. In this review, we compare the adjuvant properties of Al-NPs to those of Al-MPs in the context of infectious disease vaccines and cancer immunotherapy and provide perspectives for future research.

Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

Antibody drug conjugates: hitting the mark in pancreatic cancer?

Pancreatic cancer is one of the most common causes of cancer-related death, and the 5-year survival rate has only improved marginally over the last decade. Late detection of the disease means that in most cases the disease has advanced locally and/or metastasized, and curative surgery is not possible. Chemotherapy is still the first-line treatment however, this has only had a modest impact in improving survival, with associated toxicities. Therefore, there is an urgent need for targeted approaches to better treat pancreatic cancer, while minimizing treatment-induced side-effects. Antibody drug conjugates (ADCs) are one treatment option that could fill this gap. Here, a monoclonal antibody is used to deliver extremely potent drugs directly to the tumor site to improve on-target killing while reducing off-target toxicity. In this paper, we review the current literature for ADC targets that have been examined in vivo for treating pancreatic cancer, summarize current and on-going clinical trials using ADCs to treat pancreatic cancer and discuss potential strategies to improve their therapeutic window.

An anti-mesothelin targeting antibody drug conjugate induces pyroptosis and ignites antitumor immunity in mouse models of cancer.

BACKGROUND: Emerging evidence suggests that the mechanism of chemotherapy-induced cell death may influence the antitumor immune response in patients with cancer. Unlike immunologically silent apoptosis, pyroptosis is a lytic and inflammatory form of programmed cell death characterized by pore formation in the cell membrane and release of proinflammatory factors. Gasdermin E (GSDME) has recently gained attention after cleavage of GSDME by certain chemotherapeutics has been shown to elicit pyroptosis. This study investigated the immunomodulatory effects of a mesothelin-targeting antibody drug conjugate (ADC) in mouse models of breast and colon cancer. METHODS: The antitumor effects of the ADC were studied in EMT6 breast cancer and CT26 colon cancer syngeneic mouse models. The immunomodulatory effects of the ADC were assessed by analysis of tumor-infiltrating immune cells using flow cytometry. ADC mechanism of action was evaluated by morphology, biological assays, ADC-mediated cleavage of key effector proteins, and CRISPR/Cas9-mediated knockout (KO). Finally, the antitumor effect of ADC and Fms-like tyrosine kinase-3 ligand (Flt3L) combination therapy was evaluated in tumors expressing GSDME as well as in GSDME-silenced tumors. RESULTS: The data demonstrated that the ADC controlled tumor growth and stimulated anticancer immune responses. Investigation of the mechanism of action revealed that tubulysin, the cytotoxic payload of the ADC, induced cleavage of GSDME and elicited pyroptotic cell death in GSDME-expressing cells. Using GSDME KO, we showed that GSDME expression is critical for the effectiveness of the ADC as a monotherapy. Combining the ADC with Flt3L, a cytokine that expands dendritic cells in both lymphoid and non-lymphoid tissues, restored control of GSDME KO tumors. CONCLUSIONS: Together, these results show for the first time that tubulysin and a tubulysin containing ADC can elicit pyroptosis, and that this fiery cell death is critical for antitumor immunity and therapeutic response.

The RNA-binding protein La/SSB associates with radiation-induced DNA double-strand breaks in lung cancer cell lines.

BACKGROUND: Platinum-based chemotherapy and radiotherapy are standard treatments for non-small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment-induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies. AIM: In this study, we examined radiation-induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB. METHOD: Three lung cancer lines (A549, H460 and LL2) were irradiated with different X-ray doses or X-radiated with a 5 Gy dose and examined at different time-points post-irradiation for DNA DSB in the form of γ-H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ-H2AX co-localise and performed proximity ligation assay (PLA) and co-immunoprecipitation to confirm the interaction of these proteins. RESULTS: We found that the radio-resistant A549 cell line compared to the radio-sensitive H460 cell line showed faster resolution of radiation-induced γ-H2AX foci over time. Conversely, we found more co-localised γ-H2AX and La/SSB foci by PLA in irradiated A549 cells. CONCLUSION: The co-localisation of La/SSB with radiation-induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate this.

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®.

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anticancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug conjugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous 'eat me' signals rather than depend on functional FcγR expression for phagocytosis.

Preliminary Development and Testing of C595 Radioimmunoconjugates for Targeting MUC1 Cancer Epitopes in Pancreatic Ductal Adenocarcinoma.

Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64Cu-DOTA-C595 and 177Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM (p < 0.05). 64Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines (p > 0.05). The high cellular binding of 177Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

Characteristic differences in radiation-induced DNA damage response in human papillomavirus-negative and human papillomavirus-positive head and neck cancers with accumulation of fractional radiation dose.

BACKGROUND: Superior treatment responses by patients with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC), compared to patients with HNSCC from other causes, drive biomarker research to optimize treatment. Most HNSCC patients receive radiation therapy delivered as a fractionated course. Changing HPV status in HNSCC from a positive prognostic marker to a predictive one requires biomarkers that capture cellular radiation response to cumulative dose. METHODS: Nuclear enlargement, γH2AX expression and micronuclei count, were studied in six HNSCC cell lines after 4 Gy fractionated X-irradiation. RESULTS: All HNSCC cell lines displayed altered cellular responses, indicating increasing inability to repair radiation damage with subsequent radiation fractions. Increases in nuclear area were significantly greater among HPV positive cell lines (207% and 67% for the HPV positive and HPV negative groups, respectively). CONCLUSIONS: A different character of DNA repair dysfunction in the HPV positive group suggests greater chromosomal translocation with accumulated radiation dose.

Positron Emission Tomographic Imaging of Tumor Cell Death Using Zirconium-89-Labeled APOMAB® Following Cisplatin Chemotherapy in Lung and Ovarian Cancer Xenograft Models.

PURPOSE: Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. PROCEDURES: In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [89Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [89Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. RESULTS: Tumor uptake of [89Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. CONCLUSION: Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.

Cancer cell detection device for the diagnosis of bladder cancer from urine.

Bladder cancer is common and has one of the highest recurrence rates. Cystoscopy, the current gold standard diagnosis approach, has recently benefited from the introduction of blue light assisted photodynamic diagnostic (PDD). While blue light cystoscopy improves diagnostic sensitivity, it remains a costly and invasive approach. Here, we present a microfluidic-based platform for non-invasive diagnosis which combines the principle of PDD with whole cell immunocapture technology to detect bladder cancer cells shed in patient urine ex vivo. Initially, we demonstrate with model cell lines that our non-invasive approach achieves highly specific capture rates of bladder cancer cells based on their Epithelial Cell Adhesion Molecule expression (>90%) and detection by the intensity levels of Hexaminolevulinic Acid-induced Protoporphyrin IX fluorescence. Then, we show in a pilot study that the biosensor platform successfully discriminates histopathologically diagnosed cancer patients (n = 10) from non-cancer controls (n = 25). Our platform can support the development of a novel non-invasive diagnostic device for post treatment surveillance in patients with bladder cancer and cancer detection in patients with suspected bladder cancer.

Tumour-associated macrophages process drug and radio-conjugates of the dead tumour cell-targeting APOMAB® antibody.

APOMAB (chDAB4) is a dead tumour cell-targeting antibody which has been used preclinically as a diagnostic agent and therapeutically as a radioimmunotherapy and antibody drug conjugate (ADC). However, little is known of the intra-tumour processing of chDAB4 when bound to dead tumour cells. In this study we examine the role of macrophages in the in vitro and in vivo processing of radiolabelled chDAB4 and a chDAB4 ADC. We found that chDAB4 binds to macrophages in vitro, resulting in the killing of macrophages when using the ADC, chDAB4-SG3249. Free drug released by the macrophage processing of chDAB4-SG3249 could result in killing of 'bystander' Lewis lung (LL2) carcinoma cells. Furthermore, macrophages phagocytosed chDAB4-bound dead LL2 cells and were killed when they phagocytosed chDAB4-SG3249-bound dead LL2 cells in vitro. In vivo, we found markedly different tumour retention of chDAB4 in the LL2 tumour model depending on whether it was radiolabelled with a residualising radionuclide (89Zr), which is retained intracellularly, or a non-residualising radionuclide (124I), which can diffuse out of the cell. This prolonged retention of 89Zr vs124I indicated intra-tumoral processing of chDAB4 in vivo. The tumour uptake of 89Zr-chDAB4 was reduced after macrophage depletion, which also reduced the efficacy of the chDAB4 ADC in vivo. This study shows that macrophages can process chDAB4 and chDAB4 ADC in vitro and shows the importance of tumour-associated macrophages in the tumour retention of chDAB4 and the efficacy of chDAB4 ADC in vivo.

Intrinsic Radiosensitivity Is Not the Determining Factor in Treatment Response Differences between HPV Negative and HPV Positive Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) resulting from human papillomavirus (HPV) are increasing in incidence but demonstrate significantly better treatment response than HNSCC from other causes such as tobacco and alcohol. This study sought to identify differences in HNSCC, intrinsic to HPV status, in their response to radiation dose. Previously unexamined changes in radio-responsiveness following fractionated X-ray irradiation were compared between HPV positive and negative statuses of HNSCC. Six HNSCC cell lines, 3 of each HPV status, were investigated for radiosensitivity by clonogenic assay and modelled by response as a function of dose. Generational cultures of each cell line were developed to follow changes in radiosensitivity after repeated irradiations simulating fractionated radiation therapy. As a group, the HPV positive cell lines were more radiosensitive, but with changes following repeated fractions of dose, and modelling of response as a function of dose, both statuses displayed large radiobiological heterogeneity. These findings challenge current radiobiological assumptions of head and neck cancers as early responding tissue to radiation and may go some way in explaining difficulties reaching consensus in stratification of treatment by HPV status. Consequently, results from this study do not support stratifying radiation therapy by HPV status.

Improved non-invasive positron emission tomographic imaging of chemotherapy-induced tumor cell death using Zirconium-89-labeled APOMAB®.

PURPOSE: The chimeric monoclonal antibody (mAb) chDAB4 (APOMAB®) targets the Lupus associated (La)/Sjögren Syndrome-B (SSB) antigen, which is over-expressed in tumors but only becomes available for antibody binding in dead tumor cells. Hence, chDAB4 may be used as a novel theranostic tool to distinguish between responders and nonresponders early after chemotherapy. Here, we aimed to ascertain which positron emitter, Zirconium-89 ([89Zr]ZrIV) or Iodine-124 ([124I]I), was best suited to label chDAB4 for post-chemotherapy PET imaging of tumor-bearing mice and to determine which of two different bifunctional chelators provided optimal tumor imaging by PET using [89Zr]ZrIV-labeled chDAB4. METHODS: C57BL/6 J mice bearing subcutaneous syngeneic tumors of EL4 lymphoma were either untreated or given chemotherapy, then administered radiolabeled chDAB4 after 24 h with its biodistribution examined using PET and organ assay. We compared chDAB4 radiolabeled with [89Zr] ZrIV or [124I] I, or [89Zr]Zr-chDAB4 using either DFO-NCS or DFOSq as a chelator. RESULTS: After chemotherapy, [89Zr]Zr-chDAB4 showed higher and prolonged mean (± SD) tumor uptake of 29.5 ± 5.9 compared to 7.8 ± 1.2 for [124I] I -chDAB4. In contrast, antibody uptake in healthy tissues was not affected. Compared to DFO-NCS, DFOSq did not result in significant differences in tumor uptake of [89Zr]Zr-chDAB4 but did alter the tumor:liver ratio in treated mice 3 days after injection in favour of DFOSq (8.0 ± 1.1) compared to DFO-NCS (4.2 ± 0.7). CONCLUSION: ImmunoPET using chDAB4 radiolabeled with residualizing [89Zr] ZrIV rather than [124I] I optimized post-chemotherapy tumor uptake. Further, PET imaging characteristics were improved by DFOSq rather than DFO-NCS. Therefore, the radionuclide/chelator combination of [89Zr] ZrIV and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.

Influence of the human papillomavirus on the radio-responsiveness of cancer stem cells in head and neck cancers.

A growing proportion of head and neck cancers (HNC) result from HPV infection. Between HNC aetiological groups (HPV positive and HPV negative) clinical evidence demonstrates significantly better treatment response among HPV positive cancers. Cancer stem cells (CSCs) are identified in HNC tumour populations as agents of treatment resistance and a target for tumour control. This study examines dynamic responses in populations of a CSC phenotype in HNC cell lines following X-irradiation at therapeutic levels, and comparing between HPV statuses. Variations in CSC density between HPV groups showed no correlation with better clinical outcomes seen in the HPV positive status. CSC populations in HPV positive cell lines ranged from 1.9 to 4.8%, and 2.6 to 9.9% for HPV negative. Following 4 Gy X- irradiation however, HPV negative cell lines demonstrated more frequent and significantly greater escalation in CSC proportions, being 3-fold that of the HPV positive group at 72 hours post irradiation. CSC proportions of tumour populations are not fixed but subject to change in response to radiation at therapeutic dose levels. These findings imply a potential effect of aetiology on radio-responsiveness in CSCs, illustrating that clonogen treatment response may be more informative of therapy outcomes than inherent population density alone.

Diversity of cancer stem cells in head and neck carcinomas: The role of HPV in cancer stem cell heterogeneity, plasticity and treatment response.

Head and neck squamous cell carcinomas (HNSCC) resulting from oncogenic transformations following human papillomavirus (HPV) infection consistently demonstrate better treatment outcomes than HNSCC from other aetiologies. Squamous cell carcinoma of the oropharynx (OPSCC) shows the highest prevalence of HPV involvement at around 70-80%. While strongly prognostic, HPV status alone is not sufficient to predict therapy response or any potential dose de-escalation. Cancer stem cell (CSC) populations within these tumour types represent the most therapy-resistant cells and are the source of recurrence and metastases, setting a benchmark for tumour control. This review examines clinical and preclinical evidence of differences in response to treatment by the HPV statuses of HNSCC and the role played by CSCs in treatment resistance and their repopulation from non-CSCs. Evidence was collated from literature searches of PubMed, Scopus and Ovid for differential treatment response by HPV status and contribution by critical biomarkers including CSC fractions and chemo-radiosensitivity. While HPV and CSC are yet to fulfil promise as biomarkers of treatment response, understanding how HPV positive and negative aetiologies affect CSC response to treatment and tumour plasticity will facilitate their use for greater treatment individualisation.

APOMAB Antibody-Drug Conjugates Targeting Dead Tumor Cells are Effective In Vivo.

Antibody-drug conjugates (ADC) have revolutionized the field of cancer therapy. ADCs combine the high specificity of tumor-targeting monoclonal antibodies with potent cytotoxic drugs, which cannot be used alone because of their high toxicity. Till date, all ADCs have either targeted cell membrane proteins on tumors or the tumor vasculature and microenvironment. Here, we investigate ADCs of APOMAB (DAB4, or its chimeric derivative, chDAB4), which is a mAb targeting the La/SSB protein, which is only accessible for binding in dying or dead cancer cells. We show that DAB4-labeled dead cells are phagocytosed by macrophages, and that the apoptotic/necrotic areas within lung tumor xenografts are bound by DAB4 and are infiltrated with macrophages. We show that only DAB4-ADCs with a cleavable linker and diffusible drug are effective in two lung cancer models, particularly when given after chemotherapy. These results are consistent with other recent studies showing that direct internalization of ADCs by target cells is not essential for ADC activity because the linker can be cleaved extracellularly or through other mechanisms. Rather than targeting a tumor cell type specific antigen, DAB4-ADCs have the advantage of targeting a common trait in most solid tumors: an excess of post-apoptotic, necrotic cells either adjacent to hypoxic tumor regions or distributed more generally after cytotoxic therapy. Consequently, any antitumor effects are solely the result of bystander killing, either through internalization of the dead, ADC-bound tumor cells by macrophages, or extracellular cleavage of the ADC in the tumor microenvironment.

Experimental investigation of radiobiology in head and neck cancer cell lines as a function of HPV status, by MTT assay.

Head and neck cancers (HNCs) are aggressive epithelial tumours frequently treated using radiation. HNC biology shows distinctions dependent on the oncologic involvement of the human papilloma virus (HPV). Clinically, HPV positive HNCs respond better to radiotherapy but few in vitro data demonstrate radiobiological differences explaining differences in clinical outcomes. This pilot study examined radiobiological responses to irradiation and subsequent regeneration in two HNC cell lines (HPV positive and negative). A novel approach was taken to develop generational cultures of HNC cell lines, UM-SCC-1 (HPV negative) and UM-SCC-47 (HPV positive). MTT assays were used to determine surviving metabolic activity as a function of dose following 6 MV X-ray irradiation. Parallel cultures surviving 4 Gy irradiation (not analysed) were re-cultured and passaged to develop subsequent generations which were re-irradiated and analysed for generational change in radiation response. Second and 3rd generations of UM-SCC-1 showed decreasing metabolic activity with dose but little difference was evident in surviving fractions between these generations. Significantly lower metabolic activity in the 3rd generation at <6 Gy, compared to the 2nd generation, showed UM-SCC-47 becoming progressively more radiosensitive. HPV positive UM-SCC-47 showed generational progression in radiosensitisation not seen in the HPV negative UM-SCC-1.

In vitro investigation of head and neck cancer stem cell proportions and their changes following X-ray irradiation as a function of HPV status.

INTRODUCTION: Some head and neck squamous cell carcinomas (HNSCC) have a distinct aetiology, which depends on the presence of oncogenic human papilloma virus (HPV). Also, HNSCC contains cancer stem cells (CSCs) that have greater radioresistance and capacity to change replication dynamics in response to irradiation compared to non-clonogenic cells. Since there is limited data on CSCs in HNSCC as a function of HPV status, better understanding of their radiobiology may enable improved treatment outcome. METHODS: Baseline and post-irradiation changes in CSC proportions were investigated by flow cytometry in a HPV-negative (UM-SCC-1) and a HPV-positive (UM-SCC-47) HNSCC cell line, using fluorescent staining with CD44/ALDH markers. CSC proportions in both irradiated and unirradiated cultures were compared for the two cell lines at various times post-irradiation. To assess repopulation of CSCs, untreated cultures were depleted of CD44+/ALDH+ cells and re-cultured for 3 weeks before flow cytometry analysis. RESULTS: CSC proportions in untreated cell lines were 0.57% (UM-SCC-1) and 2.87% (UM-SCC-47). Untreated cell lines depleted of CD44+/ALDH+ repopulated this phenotype to a mean of 0.15% (UM-SCC-1) and 6.76% (UM-SCC-47). All UM-SCC-47 generations showed elevated CSC proportions after irradiation, with the most significant increase at 2 days post-irradiation. The highest elevation in UM-SCC-1 CSCs was observed at 1 day post-irradiation in the 2nd generation and at 3 days after irradiation in the 3rd generation. When measured after 10 days, only the 3rd generation of UM-SCC-1 showed elevated CSCs. CONCLUSIONS: CSC proportions in both cell lines were elevated after exposure and varied with time post irradiation. UM-SCC-47 displayed significant plasticity in repopulating the CSC phenotype in depleted cultures, which was not seen in UM-SCC-1.

Temporal Responses to X-Radiation Exposure in Spleen in the pKZ1 Mouse Recombination Assay.

The in vivo mouse transgenic pKZ1 chromosomal inversion assay is a sensitive assay that responds to very low doses of DNA-damaging agents. pKZ1 inversions are measured as the frequency of cells expressing E. coli ß-galactosidase protein, which can only be produced from an inverted pKZ1 transgene. In previous studies we reported that a single whole-body low dose of 0.01 mGy X rays alone caused an increase in pKZ1 chromosomal inversions in spleen when analyzed 3 days postirradiation, and yet this same dose could protect from high-dose-induced inversions when delivered as a conditioning dose 4 h before or after a 1 Gy challenge dose. In an attempt to explain these results, we performed temporal studies over a wide radiation dose range to determine if the inversion response was temporally different at different doses. pKZ1 mice were irradiated with a single whole-body X-ray dose of 0.01 mGy, 1 mGy or 1 Gy, and spleen sections were then analyzed for pKZ1 inversions at 7 h, 1 day or 7 days after exposure. No change in inversion frequency was observed at the 7 h time point at any dose. At day 1, an increase in inversions was observed in response to the 0.01 mGy dose, whereas a decrease in inversions below sham-treated frequency was observed for the 1 mGy dose. Inversion frequency for both doses returned to sham-treated inversion frequency by day 7. To our knowledge, this is the first reported study to examine the temporal nature of a radiation response spanning a wide dose range, including doses relevant to occupational exposure, and the results are dynamic and dose specific. The results suggest that inversions induced after low-dose irradiation are removed by homeostatic mechanisms within a short time frame, and underscore the importance of studying responses over a period of time when interpreting radiation effects.