Electrochemotherapy with bleomycin associated with doxorubicin induces tumor regression and decreases the proliferative index in canine cutaneous squamous cell carcinomas

Canine cutaneous squamous cell carcinoma (cSCC) is the most common skin cancer in dogs, and due to its low metastatic rate, local treatments such as electrochemotherapy (ECT) promote disease control or even complete remission and increase the survival time in most cases. This study aimed to evaluate the expression of BAX, Bcl-2, and Ki67 and clinical parameters in dogs with cSCC subjected to ECT. A prospective clinical nonrandomized study was performed in dogs with naturally occurring cSCC treated with ECT. Eighteen lesions (from 11 dogs) were selected, independent of breed, sex and age. The ECT protocol consisted of bleomycin plus doxorubicin followed by electric pulses characterized by 8 biphasic electric pulses lasting 100 ms, 1 Hz and 1000 V/cm. Among the 18 lesions, the lesion volume significantly decreased after treatment (p=0.04). The tumor size at D0 had no impact on survival time or prognosis (P>0.05). A decreased mitotic index was observed at compared with D0 (P=0.019). We also observed more intratumoral necrosis at D21 compared to D0 (P=0.041). The median expression level of Ki67 was 277.96 at D0 and 193.92 at D21. Thus, tumor samples had a lower proliferative index after ECT (D21) (P=0.031). The survival times of subjects with Ki67 values lower and higher than the Ki67 median value were not significantly different (P>0.05). Regarding apoptotic markers, there was no significant difference in BAX or Bcl-2 expression between D0 and D21 (P>0.05) or in overall survival between subjects with different levels of apoptotic markers. Furthermore, a positive correlation was observed between BAX and Bcl-2 before ECT (D0) (P=0.0379, r=0.5067). In conclusion, there was no change in BAX and Bcl-2 protein expression levels in response to ECT at the time points evaluated, and ECT was able to reduce tumor volume and cellular proliferation in cSCC.


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Canine cutaneous squamous cell carcinoma (cSCC) is the most common skin cancer in 56 dogs, and the most important etiological factor is chronic sunlight exposure. On the other hand, 57 canine papillomavirus infection, chronic inflammation and immunosuppression might be 58 involved in the development of cSCC [1][2][3][4]. cSCC is the second most common cutaneous tumor 59 in dogs [5], with an incidence between 3-20% [6,7]; however, the incidence of cSCC depends 60 on the geographic area [5]. In humans and dogs, actinic keratosis is an SCC precursor lesion 61 with more than 80% of the SCC cases in humans being derived from previous actinic keratosis 62 [8,9]. 63 Generally, cSCCs are locally invasive tumors with a low metastatic rate (13%) [10][11][12][13], 64 with is similar to the behavior of SCCs humans, in whom the metastatic rate is 5%; metastasis 65 mainly occurs in locoregional lymph nodes [14][15][16]. Due to this low risk of metastasis, local 66 treatments such as surgery, cryotherapy, radiotherapy, photodynamic therapy and 67 electrochemotherapy (ECT) promote disease control and increase the survival time in most 68 cases [1,17]. 69 ECT is able to induce an inflammatory response, necrosis, scar tissue and apoptosis in 70 tumors [18][19][20]. This process is a genetically programmed process for the elimination of of mammary tumors and lymphoma [25,26]. However, conflicting data have been observed 81 among the studies [25][26][27]. In feline cutaneous SCCs, neither BAX nor BCL-2 expression was 82 detected. However, in basal cell tumors, BCL-2 expression was higher (23/24) than that of 83 BAX, which was expressed only in seven out of 24 tumors. For the tumors that expressed both 84 BAX and BCL-2, the BAX:BCL-2 ratio was low [28]. 85 Another important factor to be evaluated in cSCC is cellular proliferation, which is 86 measured by the level of Ki67. It is a nuclear protein that is used to detect proliferative cells 87 because it is present in proliferating cells during the late G1, S, G2 and M phases of the cell 88 cycle, and it is correlated with poor prognosis in dogs [29][30][31][32]  Eighteen lesions (from 11 dogs) were selected, independent of breed, sex and age. The 126 eligibility inclusion criteria were as follows: dogs with clinically staged cSCC and complete 127 physical examinations, laboratory exams, and fine needle aspiration of regional lymph nodes.

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Three-view thoracic radiography and abdominal ultrasonography were also performed. of the tumor. The procedure lasted until 28 minutes, according to a previous report [42,43].

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All ECT procedures were administered under general anesthesia using propofol (5 141 mg/kg) IV followed by endotracheal intubation; anesthesia was maintained with isoflurane. All  Tumor response 146 The total volume of the neoplasm was calculated by the following formula:

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Complete remission (CR) was defined as a total reduction in measured tumor volume, while     The most commonly affected sites observed were the abdominal region (n=10), 241 followed by the thoracic region (n=5), axillar region (n=1), preputial region (n=1) and tibial 242 region (n=1). Among the 11 subjects, only three had regional lymph node involvement at the 243 time of diagnosis. There was no difference in survival time between subjects with and without 244 lymph node involvement (S Fig 1A) (P> 0.05). Among the 18 lesions, the volume before the treatment ranged from 0.14 to 112.9 cm³ 251 (median of 4.64), and the volume decreased significantly after treatment (p=0.04), ranging from 252 0.11 to 118.2 cm³ (median of 1.49) ( Table 1) (Fig 1A).  In addition, the association between the clinical stage before treatment (D0) and the 281 response to ECT was not significant (p=0.332) (Fig 1B). The median survival time of the 11 282 dogs was 180 days (32 to 570 days) (Fig 2A). The additional clinical results are shown in Table 1. The number of sessions that each 288 subject underwent ranged from one to three. Five subjects underwent a surgical procedure after 289 D21 (four due to PD and one due to the owner request).

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The median mitotic index in the tumor group at D0 was 4.94 (0 to 34). We found a 297 relationship between the mitotic index and overall survival. Subjects with mitosis numbers 298 lower than 4.9 at D0 experienced a longer survival time (P=0.009) (Fig 2B).  Additionally, we found decreased mitotic index values at D21 compared to D0 (median 310 1.5, ranging from 0 to 20) (P=0.019) (Fig 1C). We also assessed intratumoral necrosis in the 311 tumor samples at D0 and D21. It is not surprising that we found more necrosis in tumors at D21 15 312 than at D0 (P=0.041) (Fig 1D). We evaluated necrosis at D0 and the survival time, and we did 313 not find significant differences (P>0.05). expression between D0 and D21 (Fig 3 A and B). Thus, tumor samples after ECT (D21) had a 326 lower proliferative index than those before ECT (p=0.031) ( Figure 1E). Using the proliferative 327 index at D0, we evaluated the survival time between subjects with Ki67 values lower and higher 328 than the median Ki67 value, and we did not find a significant difference (P>0.05). For BAX and Bcl-2, cytoplasmic expression was identified (Fig 4A). We found 340 median BAX expression levels of 21.02% (ranging from 8.34 to 70.56) at D0 and 24.53% 341 (ranging from 10 to 74.47) at D21. There was no significant difference in BAX expression 342 between D0 and D21 (P>0.05) (Fig 4B). There was no significant difference in overall survival 343 between subjects with low and high BAX expression levels (P>0.05) (Suppl Fig 1C).  Regarding Bcl-2 expression (Fig 4C), we found medians of 20.62% (ranging from 355 2.76% to 48.37) at D0 and 26.42% at D21 (ranging from 0.65 to 77.23). There was no 356 statistically significant difference in Bcl-2 expression between D0 and D21 (P>0.05) (Fig 4D).

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When we evaluated the survival time between samples with low Bcl-2 expression versus those 358 with high expression, we did not find a significant difference (P>0.05) (Suppl Fig 1D).

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Furthermore, a positive correlation was observed between BAX/Bcl-2 expression levels 360 before ECT (D0) (p=0.0379, r=0.5067) (Fig 4E). However, after treatment (D21), we did not 17 361 find a significant Gaussian approximation (p=0.7370, r=0.0911) (Fig 4F). Regarding the 362 correlation between BAX/Ki67 and Bcl-2/Ki67, there was no significant difference among 363 them at different time points (p>0.05). Western blot analysis 372 We found a 26-kDa band for Bcl-2 and a 21-kDa band size for BAX. Both antibodies 373 showed only one band each in the western blot analysis (Fig 5).  Interestingly, lymph node involvement was not a prognostic factor in dogs with cSCC.

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The number and size of the lesions seems to be more important than the involvement of local