Prevention of in Situ Recurrence and Distant Metastasis in Feline Mammary Carcinoma Treated with Plasmonic Photothermal Therapy

doi:10.21203/rs.3.rs-2215572/v1

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Prevention of in Situ Recurrence and Distant Metastasis in Feline Mammary Carcinoma Treated with Plasmonic Photothermal Therapy

https://doi.org/10.21203/rs.3.rs-2215572/v1

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Background

Clinical studies indicated the role of Breast Cancer Stem Cells (BCSCs) with the phenotype of CD44⁺/CD24⁻ and/or CD133⁺ in the promotion of invasive and metastatic mammary tumors. Where BCSCs are resistant against the conventional chemo and radiotherapies, the gold nanorods (AuNRs)-mediated plasmonic photothermal therapy (PPTT) selectively eradicated BCSCs. A change in CTCs number predicts response to therapy. Among felines, most of the queens with mammary carcinoma (MC) exhibit an aggressive metastatic phenotype. Thus, indicated the importance of developing a rapid, accurate and noninvasive approach for prediction and monitoring of metastasis in cats. In this study, we investigated for the first time the anti-tumor activity of AuNRs PPTT by Flow Cytometry Analysis (FCA) of the circulatory BCSCs in cats with spontaneous metastatic and non-metastatic forms of MC.

Methods

The PPTT was applied on each animal in this investigation either alone (GA) or in combination with mastectomy (GB). PB was collected from all diseased cats before the treatment and 2 weeks after every PPTT cycle. BCSCs were identified in each sample as CD133⁺ in one test and CD44⁺/CD24⁻ combination in another test using the FCA. Cells counts were compared between groups and across time. The correlation between BCSCs values and metastasis was evaluated using Spearman's correlation test. Survival analysis was performed, and prognostic cutoff points were established using ROC analysis.

Results

With survival rate of 50%, the non-metastatic cats in GA revealed complete tumor ablation 6–8 weeks of treatment and complete wound healing in GB, no in situ recurrence or distant metastasis over the observation period with a statistically significant drop in cBCSCs counts (P = 0.00512). Progression-free survival (PFS) was of a mean of 9 months (GA) and 6.6 months (GB) with median overall survival (OS) of 10.5 months (GA) and 9.5 months (GB). Lung metastasis persisted in metastatic cats in GA &GB with short PFS of 4 months. Prognostic significant cutoff values were > 996 for (CD44⁺/CD24⁻) phenotypes and > 110 for CD133⁺ cells.

Conclusion

BCSCs are highly susceptible to PPTT in non-metastatic queens unlike the metastatic cats as a treatment modification is required to systemically deliver the AuNRs to the metastatic sites. FC enumeration of cTCs with the CD44⁺/CD24⁻ or CD133⁺ phenotypes has a prognostic value as an accurate and non-invasive method for therapy monitoring in FMC.

Feline Mammary Tumor

Gold Nano-Rods

Plasmonic Photothermal Therapy

Flow Cytometry Analysis

Prognosis

Metastasis

The third common type of malignancy in cats is spontaneous feline mammary carcinoma (FMC). Nearly 90% of these tumors are malignant, extremely aggressive with rapid metastatic potentials (1). Numerous characteristics support the use of cats as a compatible animal model for human breast cancer (HBC) research, including the fact that these species share many environmental risk factors with humans, unlike typical laboratory animals. Besides, the cancer related genes showed greater homology between cats and humans than between humans and other animals (2, 3). Furthermore, cats have a shorter lifespan and rapid cancer progression that ensure a fast trail completion and data collection (4).

Since Al-Hajj and colleagues were successful in isolating and identifying tumorigenic cell subpopulations in breast cancer patients (5), most studies have demonstrated the role of Tumor Initiating Cells (TIC) or Cancer Stem Cells (CSC) in breast cancer recurrence and metastasis. Breast cancer stem cells (BCSCs) with CD44⁺/CD24⁻ and/or CD133⁺ phenotypes have been linked to increased invasive and metastatic potential (6–10). On the level of companion animals, much research has reported the isolation and characterization of BCSC in feline (11–13) as well as in canines (14–16). These isolated cells showed self-renewal, proliferative and tumor initiative capabilities.

Through a process known by Epithelial- Mesenchymal Transition (EMT), some tumor cells (known by Circulating Tumor Cells, CTCs) gain their ability to intravasate to the surrounding blood or lymphatic vessels, survive in the circulation or lymphatic system and finally extravasate to the distant sites (17). During the EMT, CTCs lose their epithelial characteristics (cell adhesion and cuboid shape) and gain mesenchymal traits (motility and elongated spindle shape) by downregulation of some surface biomarkers and upregulation of another (E-cadherin, CD44v and N-cadherin, CD44s, and vimentin respectively) (18). A subpopulation of CTCs acquired stemness characters during EMT (as self-renewal and proliferation), these are the circulating Cancer Stem Cells (cCSCs) (19).

By forming tumor micro emboli and platelets mixed aggregates, CTCs and their subpopulation cCSCs can escape lysis by natural killer (NK) cells, survive in the bloodstream and resist apoptosis which enhances their metastatic capabilities (20–22), and thus explained why many studies introduced enumeration of CTCs and cCSCs as highly informative liquid biopsy with prognostic value (23).

Recently, serial analysis of CTCs and cCSCs in Peripheral Blood (PB) has been demonstrated to be an excellent non-invasive and easily accessible technique for the prediction, diagnosis, and prognosis of metastatic breast cancer (24). Being limited to live tumor status, CTCs and cCSCs have superiority over the other blood biomarkers as they represent active tumor spots (25)

C. Lee and colleagues discovered cCSCs to be an independent prognostic factor for patients with metastatic BC in a study. They mentioned a cutoff point of 13.3/mL CTCs and a 32.5% cCSCs with a Progression Free Survival (PFS) of 5.7 months for patients that showed high numbers of cCSCs (26). In another earlier research in non-metastatic patients, a median cutoff point of 3.4 CTCs and 23% cCSCs per 5mL PB were reported. The patients who expressed a high cCSCs count at the base line were of lower PFS after chemotherapy (18.8%) or surgery and radiotherapy (16.7%) than those with fewer cCSCs (87.1%) count in the same study (27).

Among veterinary oncology research, few studies have pointed to the prognostic and diagnostic impact of CTCs and cCSCs. In a recent study, the authors detected at least 1 CTC per 7.5 mL of PB in 44.4% of the evaluated samples from dogs with metastatic mammary carcinoma which was significantly correlated with poor outcomes (16). On the level of feline, our team has published lately a preliminary first of kind study that assessed the impact of Flow Cytometry Analysis (FCA) of cCSCs in cats PB and established a cutoff point of > 276 for CD44⁺/CD24⁻ cells and > 12 for CD133⁺ populations (28). The correlation of CTCs and cCSCs feline mammary tumor outcomes are not studied yet.

As TICs showed high resistance against the conventional chemo and radiotherapies (12, 29, 30), the need for new treatment procedures targeting CSCs is crucial. Nanomaterials were introduced as a novel treatment approach in cancer therapies. Different forms, sizes and modifications of nanomaterials (noble metals, carbon, DNA and organic polymers) are easily prepared and represent a promising approach for targeting the CSCs (31). Many scientists have evaluated the gold-based nanoparticles in association with thermal therapy as a potential targeting therapy of BCSCs (32). Xu et al. have described how the gold nanorods (AuNRs)-mediated photothermal therapy (PTT) selectively eradicated BCSCs. They mentioned that AuNRs were rapidly assumed by the BCSCs and noticed a reduction of the ALDH⁺ cells and retardation in the ability of the mammosphere formation by the treated cells (33).

Many former investigations have approved the inhibitory role of PTT and gold nanoparticles on the BC either at the primary tumor sites or on the early metastasis at the lymph nodes (34–36). In pets, Ali et al. were the first to apply the Plasmonic PTT in association with AuNRs for the treatment of feline and canine mammary carcinoma. They used three PPTT treatment sessions, two weeks apart, to gradually suppress the natural tumors in dogs and cats under study. Even one year after treatment, no relapse or toxicity was observed in any of the subjects who were exposed to AuNRs-PPTT (37). The same team has developed an effective treatment of large tumors (≥ 10cm), by combining both PPTT and surgical resection. This regime showed complete remission without any recurrence for1-2 years post application (38).

In this study, we aimed to investigate the effect of AuNRs-PPTT on cBCSCs in metastatic and non-metastatic forms of mammary tumors in cats, and to estimate the FC enumeration of cCSCs as an independent monitoring and prognostic factor in feline breast cancer.

2.1. Animals’ groups:

Ten client-owned, adult, unspayed female cats were admitted to the referral animal hospital of the Faculty of Veterinary Medicine at Cairo University. The queens were of different breeds and aged between 8–19 years old. All the cats were diagnosed with mammary carcinoma without any other disease conditions, and their owners claimed that their animals did not receive any treatment before they arrived at the clinic.

We followed the modified World Health Organization grading system to determine the primary tumor size and the presence of lung metastasis (39)

Animals were divided into 2 groups according to the tumor size; Cats with tumor size < 3cm diameter (GA = 7) were introduced to PPTT alone, while those of tumors > 3cm diameter (GB = 3) were treated by combination of surgery (mastectomy) followed by adjuvant therapy with PPTT to the tumor bed and wound margin (Table 1).

through radiography; 3 queens in GA showed lung metastasis either before (n = 2) or during (n = 1) treatment course, while in GB; only 1 cat has been diagnosed with lung metastasis before the treatment starts (Table 1).

Table (1): Descriptions of animal groups.

Group name	No. of Cases	X-ray metastasis	MT description	Treatment regime
GA	4	Free	Cats diagnosed with mammary carcinoma (< 3cm diameter)	PPTT only
	1	Developed metastasis during treatment
	2	Showed metastasis before treatment
GB	2	Free	Cats diagnosed with mammary carcinoma (> 3cm diameter)	Mastectomy followed by PPTT to the tumor bed and wound margins
GB	1	Showed metastasis before treatment	Cats diagnosed with mammary carcinoma (> 3cm diameter)

2.2. Gold Nano Rods (AuNRs) and PPTT application:

AuNRs and 808 nm diode laser (with a power of 0.5 W/cm2, and a spot size of around 5.6 mm2) were Kindly gifted by Prof. Dr. Mostafa A. El-Sayed; Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, USA. Gold nanorods were synthesized and characterized according to the previously reported method (40). The final characterization of AuNRs was with an average size of 27(± 5) ×6 (± 1) nm (length×width) and conjugated with polyethylene glycol (PEG) thiol and Arg − Gly − Asp (RGD) peptides surface ligands. The effective dose of AuNRs solution and method of implementation was adjusted according to Ali et al (37, 38).

We applied PPTT on each animal in this investigation either alone (GA = 7) or in combination with mastectomy (GB = 3). The surgeons' team at the Department of Surgery and Anaesthesiology in the Faculty of Veterinary Medicine, Cairo University proceeded with mastectomy operation as described before (41). The peri and post-operative measures were performed according to AAHA/AAFP pain management guidelines for dogs and cats (42)

All the cats in G.A were subjected to one or more PPTT cycles each 2 weeks until complete tumor ablation while in GB only one PPTT session was applied to the tumor bed after mastectomy.

2.3. Blood sampling:

Whole blood samples were collected from every female cats in this study (GA and GB) at different time points; 1ml whole blood from all diseased cats before treatment (baseline sample) and 1ml whole PB from GA and GB queens 2 weeks after every PPTT treatment cycle. All samples were stored at 4C° till analysis for a maximum of 72hrs post collection (43). Rapid blood sample processing is preferable when possible.

2.4. Flow cytometry analysis:

The entire PB samples were examined using a flow cytometer (Coulter Epics XL, Beckman Coulter, CA) to count Circulatory Breast Cancer Stem Cells (cBCSCs), and the data were analyzed using Flow Jo software (BD). According to the manufacturing structure, cBCSCs were identified in each sample using the following specific cell surface markers: CD133⁺ in one test using (CD133/1 (AC133) Antibody, Anti-Human, PE, REAlease, Miltenyi Biotec Germany), and CD44⁺/CD24^- combination in another test using (PE Mouse Anti-Human CD44v6 (2F10) BD Biosciences, USA and FITC Mouse Anti-Human CD24 (ML5), BD Bioscience). Since CD44v6 is almost completely absent from the majority of leukocytes and has been shown in studies to be a prognostic indicator for BC (18, 44, 45), we utilized CD44v6 antibody.

The used anti-human antibodies have been previously reported to stain feline samples by FCA (12, 13, 28). One tube served as a negative control (unstained cells) for each sample. Following labelling with particular antibodies, RBC were lysed and peripheral blood leukocytes were fixed in accordance with the Thermo Fisher Scientific flow cytometry methodology for staining cell surface antigens, which was covered in our previously published study (28)

Due of the sporadic nature of cBCSCs, we used the gating approach previously discussed (28), and recorded every event in the whole suspension. The cBCSCs were recognized as CD133⁺ or CD44⁺/CD24^- combination.

2.5. Statistical analysis:

Normality was checked using the Shapiro Wilk test, box plots, and descriptive. CD44⁺/Cd24^- and CD133⁺ values were presented mainly using Median, Inter Quartile range, minimum and maximum values due to the non-normal distribution of the data. Cells count values were compared between groups using the Mann Whitney U test and for comparisons across time, the Wilcoxon Sign Rank test was applied. The correlation between values of CTCs bearing CD44⁺/CD24^- and CD133⁺ stem cell markers and metastasis at metastatic animals was done using Spearman's rank correlation coefficient test. Survival analysis was performed using Kaplan Meier Estimator based on the outcome “Dead”. Area Under Curve, sensitivity, specificity, and cutoff points were calculated. Sensitivity values were plotted against complementary specificity values in the receiver operating characteristic (ROC) curve. The significance level was set at a p-value of 0.05. We used MedCalc Statistical Software version 19.2.6 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org;2020) and SPSS (windows version 23) for data analysis.

3.1. The treatment responses of the PPTT regimes:

All the cats under this investigation were subjected to either PPTT alone (GA = 7) or the PPT combination therapy with mastectomy (GB = 3).

Four GA animals demonstrated complete tumor ablation within 6–8 weeks of treatment without the occurrence of in situ recurrence or distant metastasis over the course of the observation period. This was correlated with progression-free survival (PFS) of a mean of 9 months and prolonged overall survival (OS), which in some cases reached more than 25 months (Fig. 1&2).

Another 2 metastatic queens in the same group (GA) showed partial tumor remission with lung metastasis persisting. In addition, a nonmetastatic cat in the GA underwent treatment and, with a short OS of three months, acquired lung metastases and additional mammary cancers.

Two non-metastatic mammary tumor-bearing cats in GB showed complete wound healing two weeks after surgery without the development of in situ recurrence or distant metastasis, while a metastatic queen in the same group showed complete wound healing two weeks after surgery with persistence of the lung metastasis and a low PFS of 3 months (Fig. 1&2).

3.2. Evaluation of the prognostic ability of the designed FCA protocol:

The ROC study for the ability of the Flow Cytometry Analysis to discriminate between metastatic and non-metastatic cases (Table.2) showed an excellent AUC value for both CD44^+/CD24⁻ and CD133⁺ phenotypes (excellent Ref. ratio between 0.9 and 1) with a statistically significant P-value (< 0.0001) for both phenotypes (Fig. 3). The obtained prognostic cut-off points of (> 996) for CD44^+/CD24⁻ cells and (> 110) for CD133⁺ cells generate the high Youden index of 1.00 and 0.75 respectively.

Table (2): AUC for CTCs bearing CD44 ⁺ /CD24 ⁻ and CD133⁺ stem cell markers among diseased cases (Prognostic ability).

		CD44⁺/CD24⁻	CD133⁺
AUC	Estimate	1.000	0.917
	95% CI	(0.692 1.000)	(0.575–0.999)
	P-value	< 0.0001^*	< 0.0001^*
Sensitivity		100.00	75.00
Specificity		100.00	100.00
Youden Index		1.0000	0.7500
Cutoff Point		> 996	> 110
*Statistically significant at p value ≤ 0.05

3.3. Prognostic relevance of cBCSCs CD44⁺/CD24⁻ and CD133⁺ subpopulation before and after treatment:

The non-metastatic felines had a statistically significant drop in the CD44⁺/CD24⁻ and CD133⁺ phenotypic values after the treatment with PPTT alone (GA) when compared either to the base line values (P = 0.00512) or the metastatic animals in the same group (p-value = 0.008) (Table.3) (Fig. 4 &5).

In contrast, non-metastatic animals treated with combination therapy (GB, n = 2) showed a notable drop in the number of cTCs with the CD44⁺/CD24⁻ and CD133⁺ phenotypes (Table.3) (Fig. 4 &5), as opposed to the one metastatic instance in the same group that showed a striking rise in these phenotypes (Table 3).

The Spearman's rank correlation test indicated a strong statistically significant correlation (p = 0.005) between cells bearing CD44⁺/CD24⁻ markers values and metastasis with (RHO = 0.943), unlike CD133⁺ values that were of weak correlation (Table.4).

Table (3): Monitoring of PPTT therapeutic efficacy through enumeration of cBCCs bearing (CD133 ⁺ and CD44⁺/CD24⁻) stem cell markers using FCA. Results represented by Median.

Groups	No. Of cBCSCs per 1ml blood			Fold change
Groups	Marker	Before	After	Fold change
GA: Non-metastatic	CD44⁺/CD24⁻	8.964× 10³	0.296 × 10³	-0.96724^*
GA: Non-metastatic	CD133⁺	0.101 × 10³	0.027 × 10³	− 0.94382^*
GA: metastatic	CD44⁺/CD24⁻	0.026× 10³	62.043× 10³	2385.269
GA: metastatic	CD133⁺	0.162× 10³	2.544× 10³	14.7037
GB: Non-metastatic	CD44⁺/CD24⁻	9.416× 10³	0.495× 10³	-0.94743^*
GB: Non-metastatic	CD133⁺	1.093× 10³	0.012× 10³	− 0.98902^*
GB: metastatic	CD44⁺/CD24⁻	5.932× 10³	21.323× 10³	2.59
GB: metastatic	CD133⁺	0.286× 10³	3.728× 10³	12.03
^* Negative fold change: Indicates downregulation of cBCCs bearing (CD133⁺ and CD44⁺/CD24⁻) stem cell markers after treatment with PPTT.

Table (4): Correlation between values of CTCs bearing CD44⁺/CD24^- and CD133⁺ stem cell markers and metastasis.

Spearman’s ρ	CD44⁺/Cd24⁻	CD133⁺
Rho	0.943	-0.20
P- value	0.005*	0.704
*Statistically significant at p value ≤ 0.05.

3.4. Survival analysis:

During a total follow-up period of 29 months (March 2019-August 2021), the recruited queens showed a 50% survival rate (Table 5). The cats of GA and GB showed a median of 10.5 and 9.5 months respectively overall survival (OS) and progression-free survival (PFS) of a mean of 9 and 6.6 months respectively, while metastatic cat had a median OS of 15.5 months and PFS of a mean of 4 months (Fig. 1&2).

Table (5): Total survival rate of GA and GB at the end of the study

Groups		GA (n = 7)	GB (n = 3)	Total
Survival: n (%)	live	4 (57%)	1 (33.3%)	5 (50%)
Survival: n (%)	Dead	3 (42.8%)	2 (66.6%)	5 (50%)

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 (46, 47), 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 (12, 16, 28, 48, 49)

We established prognostic significant 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, 50).

According to Figueira et al (51) the presence of neoplastic emboli, upregulation of P-Cadherin, and loss of expression or abnormal function of E-Cadherin increase tumor cell proliferation, motility, invasiveness, high infiltrative growth, and consequently increase CTCs and cCSC populations in queens with mammary tumors, which may account for the major 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 (52) 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, 53, 54).

Besides, factors such as gold nanoparticles' (GNPs') anti-angiogenic property, which inhibits the function of pro-angiogenic heparin-binding growth factors (HB-GFs) (55), 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, 56, 57) 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, 58, 59). 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 (60) 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) (61) 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 (61)

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 (62) 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 (63, 64) 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 (65)

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 researches that confirmed the CD44⁺/CD24^- cells as a prognostic metastatic marker at HBC (26, 30, 56) 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 (66) or with dogs in another investigation (16) El-Rasikh et al (67) 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)(67). This vast difference could be because of our relatively long follow-up period (up to 29 months).

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).

ADCC	Antibody Dependent Cytotoxic Cells
AUC	Area under the curve
AuNRS	Gold nano rods
BCSCs	Breast cancer stem cells
cBCSCs	Circulating breast cancer stem cells
CMT	Canine mammary tumor
CSC	Cancer stem cells
cTCS	Circulating tumor cells
CTLs	Cytotoxic T Lymphocytes
ctDNA	Circulating tumor DNA
EMT	Epithelial mesenchymal transition
EpCAM	Epithelial cell adhesion molecule
FACS	Fluorescence‐activated cell sorting
FCA	Flow cytometry analysis
FMC	Feline mammary carcinoma
FMTs	Feline mammary tumors
GNPs'	Gold Nano Particles’
HBC	Human breast cancer
HB-GFs	Heparin-Binding Growth Factors
ICD	Immunogenic Cell Death
MET	Mesenchymal epithelial transition
NIR	Near infra-red
PB	Peripheral blood
PPTT	Plasmonic photo thermal therapy
ROC	Receiver operating characteristic
TICs	Tumor-initiating cells
WHO	World health organization

Ethics approval and Consent of Owners: This study was carried out in compliance with the ARRIVE guidelines (68,69) and Studies of diagnostic accuracy (STARD) guidelines (70). All animals under investigation in the study were handled following the Association for Assessment and Accreditation of Laboratory Animal Care and Office of Laboratory Animal Welfare (AAALAC) guidelines under the direction of the Institutional Animal Care and Use Committee at Cairo University (CU-IACUC) that approved the protocol (code: CU II F 9 16) renewed by (Vet CU28/04/2021/266). Written informed consent was provided by the owners of the cats for consideration in our research.

Consent of publication: Not applicable.

Compete of Interest: No potential competing of interest was reported by authors.

Data Availability: Data are available from the corresponding author on reasonable request.

Funding: This study was supported by the Egyptian Expatriates and Scientific Organization's joint collaborative efforts to address national R&D challenges (JESOR), ID: 34.

Author Contribution: Rasha H. Elsabagh: Validation, Investigation, Resources, Data Curation, Writing - Original Draft, Visualization, Funding acquisition, Haithem A. M. Farghali: Methodology, Validation, Resources, Writing - Review & Editing, Funding acquisition, Supervision, Ibrahim A. Emam: Investigation, Data Curation, Resources, Hams Abdelrahman: Statistical analysis, editing, Eman Ragab: Supervision, Abdelfattah A. Nada: Supervision, and Salah. A. Selim: Conceptualization, Methodology, Writing - Review & Editing, Supervision, Project administration.

Acknowledgments: We thank Prof. Dr. Mostafa A. El-Sayed; (Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, USA) for kindly providing us with AuNRs and 808 nm diode laser. We thank Christopher P Cook and Aymen Khalifa (Department of Dermatology, University of California, San Francisco, San Francisco CA, USA) for their generous contribution in editing of flow cytometry analysis.

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Prevention of in Situ Recurrence and Distant Metastasis in Feline Mammary Carcinoma Treated with Plasmonic Photothermal Therapy

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction:

2. Materials And Methods:

2.1. Animals’ groups:

2.2. Gold Nano Rods (AuNRs) and PPTT application:

2.3. Blood sampling:

2.4. Flow cytometry analysis:

2.5. Statistical analysis:

3. Results:

3.1. The treatment responses of the PPTT regimes:

3.2. Evaluation of the prognostic ability of the designed FCA protocol:

3.3. Prognostic relevance of cBCSCs CD44⁺/CD24⁻ and CD133⁺ subpopulation before and after treatment:

3.4. Survival analysis:

4. Discussion:

5. Conclusion:

List Of Abbreviation:

Declarations:

References:

Additional Declarations

Status:

Version 1

Prevention of in Situ Recurrence and Distant Metastasis in Feline Mammary Carcinoma Treated with Plasmonic Photothermal Therapy

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction:

2. Materials And Methods:

2.1. Animals’ groups:

2.2. Gold Nano Rods (AuNRs) and PPTT application:

2.3. Blood sampling:

2.4. Flow cytometry analysis:

2.5. Statistical analysis:

3. Results:

3.1. The treatment responses of the PPTT regimes:

3.2. Evaluation of the prognostic ability of the designed FCA protocol:

3.3. Prognostic relevance of cBCSCs CD44+/CD24− and CD133+ subpopulation before and after treatment:

3.4. Survival analysis:

4. Discussion:

5. Conclusion:

List Of Abbreviation:

Declarations:

References:

Additional Declarations

Status:

Version 1

3.3. Prognostic relevance of cBCSCs CD44⁺/CD24⁻ and CD133⁺ subpopulation before and after treatment: