Plasmonic Photothermal Therapy (PPTT) in FMC is a promising, alternative or supplement to the conventional cancer treatment approaches as it provides rapid recovery and less complications [37, 38].
In this study we applied the FCA to the cat’s peripheral blood as a prediction and monitoring tool to track the impact of AuNRs PPTT on the circulatory mammary tumor cells expressing stem cell markers (CD44+/CD24− population and CD13+ cells).
In an initial analysis [28], researchers described a modified FCA technique as a fast, sensitive, and specific tool for the diagnosis of FMC by detection and enumeration of cBCSCs. In our interrogation, we confirmed the prognostic ability of the previously mentioned modified FCA method and indicated its ability to distinguish between metastatic and non-metastatic animals in our research.
The (CD44+/CD24−) and CD133+ cell populations were detected in the PB of all animals under investigation with a sensitivity of 100% for (CD44+/CD24−) population and 75% for CD133+ cells, a specificity of 100% and an excellent AUC value for both phenotypes. That’s strongly supported the former evidence of the sensitivity of FC quantifications of the circulating and disseminated tumor cells at preclinical models of metastasis [49, 50], and presented the FC enumeration of cBCSCs as an accurate and non-invasive method for prognosis and therapy monitoring in feline mammary carcinoma, as well as breast cancer in both humans [25–27, 29, 30] and companion animals [13, 16, 28, 51, 52].
We established prognostic significant (P = 0.0001) cutoff values for the measurements of cBCSCs in 1 mL of feline peripheral blood for the first time in cats. These values are > 996 for (CD44+/CD24−) phenotypes and > 110 for CD133+ cells, compared to humans, whose prognostic cutoff thresholds of 2CTCs/4ml blood and 3CTCs/1ml blood have been observed in different studies [25, 53].
In cats with mammary tumors, Figueira et al [54] found that the presence of neoplastic emboli, the elevation of P-Cadherin, and loss of expression or improper function of E-Cadherin promote tumor cell proliferation, motility, invasiveness with excessive infiltrative growth and therefore, expansion of CTCs and cCSC populations. This may explain the remarkable differences between cat and human prognostic cutoff values. The non-metastatic animals in both GA(n = 4) and GB(n = 2) showed a statistically significant drop in the counts of cBCSCs by FCA (P = 0.00512) that reached its previously indicated normal values or below [28]. This retardation was accompanied by an excellent response to the PPTT, as all treated tumors in GA entirely disappeared with complete wound healing in animals at GB after 6 to 8 weeks of the administration of the treatment, without any in situ recurrence or metastasis, which supported the findings by Ali et al[37, 38] who were the first to use this technology in the treatment of cat's mammary cancers.
Furthermore, this reduction was consistent with earlier research that demonstrated the damaging effects of AuNRs on breast cancer stem cells [31, 33] and prostate CSC [54] and supported the prior articles that indicated the role of PPTT alone or in combination with some NPs to combat the onset of cancer metastasis at tumor site or in its early stages in the associated lymph node [36, 55, 56].
Besides, factors such as gold nanoparticles' (GNPs') anti-angiogenic property, which inhibits the function of pro-angiogenic heparin-binding growth factors (HB-GFs) [57], the damaging effect of hyperthermia on blood vessels, and the reported killing abilities of AuNRs [31, 33] on BCSCs could prevent BCSC residuals from escaping into the tumor bed or the wound margin during mastectomy. That explains the informed decrease in cBCSCs numbers in our study.
The other metastatic queens in the GA (n = 3) and the other one in GB exhibited a striking non-significant increase in the cBCSCs count by the FCA between the baseline and the last sample rates that accompanied by partial tumor remission and /or developing new tumors with persistence of lung metastasis.
Many former studies have confirmed the correlation between cBCSCs and progression of metastasis, tumor aggressive forms and treatment resistance [5, 58, 59] which interprets the elevated counts of cBCSCs. Also, the cancerous cells undergo Epithelial-Mesenchymal Transition (EMT) during the early stages of metastasis, which allows them to acquire stemness characteristics in addition to migrant and invasive abilities that aid in their escape from the primary tumor sites, intravasate into the circulation, and then extravasate at distant secondary tumor sites [17, 60, 61]. This fact suggests that the CSCs were already present in the circulation of the metastatic cats prior to the beginning of AuNRs PPTT.
Moreover, the localized nature of the applied treatment: the effective dose of AuNRs solution was injected inside the tumor mass, and laser irradiation was limited to the tumor site [37] rules out any possibility of the PPTT having a systematic effect on the cBCSCs or lung metastasis.
The capacity of AuNRs-PPTT to trigger apoptosis has previously been revealed in a study by Ali et al [62] who found that the AuNRs-PPTT had a significant molecular influence on both apoptosis and NETosis pathway. This effect was attributed to the cytochrome c and p53-related apoptotic pathways. This type of immunogenic cell death (ICD) will then result in the release of endogenous damage-associated molecular patterns (DAMPs) and chemokines, which help prime the immune system by activating dendritic cells (DCs) [63]. This enhances antigen presentation to T cells and stimulates the production of cytotoxic T lymphocytes (CTLs) and antibody dependent cytotoxic cells (ADCC) beside normalizing tumor vasculature [63].
This demonstrates the local damage effect of PPTT on BCSCs and other malignant cells in situ, as well as the eradication of circulatory BCSCs via abscopal effect in non-metastatic animals. In addition, it explains the statistically significant difference (P = 0.008) in the median numbers of cBCSCs populations (CD44+/CD24− and CD133+) between metastatic and non-metastatic cases following the end of treatment. Besides, the higher PFS mean (9 months) in non-metastatic cats than that of the metastatic ones (PFS: 4.16 months).
This enormous rise of the median count of the cBCSCs cells post-treatment in metastatic cats disproves the hypothesis that the abscopal effect might have any influence on distant metastasis and go in hand with Jatoi et al [64] who reported that there is no evidence that the locoregional treatment has any impact on distant metastasis and survival outcomes. In Addition, it is supporting the demonstrations of Kaplan et al. who mentioned that the primary tumors prepare the distant organs microenvironment for tumor settlement even before the arrival of CSCs. These measurements facilitate CSCs migration, survival in circulation and increase their resistant to treatments [65, 66]. Furthermore, the high burden of cBCSCs in the circulation or metastatic sites with a relatively weak systematic immune response to eradicate such high numbers of cBCSCs may be considered for the absence of the abscopal effect in the metastatic queens, even though the mechanism of inhibition of the abscopal effect in metastatic patients is still unknown [67]. In our interrogation, the metastatic queens presented a strong statistically significant (p = 0.005) correlation between values of CD44+/CD24− cells and metastasis with (RHO = 0.943). These results strongly support the finding of previous research that confirmed the CD44+/CD24− cells as a prognostic metastatic marker at HBC 26,30,58 and associated with poor outcomes in CMT [16]. In contrast to CD44+/CD24− cells, CD133+ populations showed a weak correlation with metastasis. According to previous research, there is a link between tumor aggressiveness and evolution (rather than metastasis) and expansion in CD133 expression [5].
The recruited cats in this analysis showed a relatively high survival rate of 50% when compared with queens that underwent different treatment procedures [68] or with dogs in another investigation [ 16]. El-Rasikh et al [69] were the first to apply survival analysis to cats treated with AuNRs PPTT. They mentioned an OS of a median of 17.6 and 10.23 months at nonmetastatic animals treated either with AuNRs PPTT only or in combination with surgery respectively while our OS analysis of GA and GB queens showed a median of 10.5 and 9.5 months respectively.
The metastatic cats in our study revealed a considerably higher OS of a median of 15.5 months than that of the metastatic group mentioned by El-Rasikh (3.7months)[69]. This vast difference could be because of our relatively long follow-up period (up to 29 months).
To Summarize, in the presented interrogation the FC enumeration of cTCs bearing the CD44+/CD24- or CD133+ phenotypes were confirmed as an accurate and non-invasive method for prognosis and therapy monitoring in feline mammary carcinoma, with prognostic significant cutoff values of > 996 for (CD44+/CD24-) phenotypes and > 110 for CD133+ cells. BCSCs are highly susceptible to PPTT, as demonstrated by the abscopal effect in the non-metastatic cats with mammary carcinoma. While in metastatic animals, a modified PPTT is advised to overcome the main challenge of delivering AuNRs to metastatic sites.
One of this study’s restrictions; is that we cannot apply a single flow test including CD44+, CD24-, and CD133+ as the available CD44 and the CD133 antibodies were of the same fluorophore (PE).