Enabling Chemo-Immunotherapy with HIFU in Canine Cancer Patients

Abstract

High intensity focused ultrasound (HIFU) is a promising non-invasive technique for treating solid tumors using thermal and histotripsy-based mechanical ablation. However, its clinical significance in different tumor types is not fully understood. To assess its therapeutic efficacy and immunomodulatory properties, we compared HIFU thermal ablation and histotripsy ablation in dogs with spontaneous tumors. We also evaluated the ability of non-ablative HIFU-based mild hyperthermia (40–45 °C) to improve Doxorubicin delivery and immunomodulation. Our results showed that HIFU thermal ablation induced tumor remission in the majority of treated patients over 60 days, while histotripsy achieved partial response to stable disease persistence. The adverse effects of thermal ablation were minor to moderate, while histotripsy exposures were relatively well-tolerated. Furthermore, we observed a correlation between HIFU-therapeutic response and serum anti-tumor cytokine profiles and the presence of functionally active cytotoxic immune cells in patients. Similarly, Doxorubicin-treated patients showed improved drug delivery, efficacy, and anti-tumor immune responses with HIFU hyperthermia. In conclusion, our study demonstrates that depending on the tumor type and treatment parameters, HIFU treatments can enable tumor growth control, immune activation, and chemotherapy in veterinary patient. These findings have significant clinical implications and highlight the potential of HIFU as a promising cancer treatment approach.

Introduction

High-intensity focused ultrasound (HIFU) is a non-invasive and non-ionizing treatment modality that can be used to induce solid tumor remission, drug delivery, and immune activations at various locations in the body [1, 2]. Various types of HIFU treatments can achieve distinct tumor responses, including those that employ thermal and histotripsy-based mechanical ablation, as well as mild hyperthermia for enhanced drug delivery [3–7]. HIFU thermal ablation uses high duty cycle pulsed waves to generate heat and destroy targeted tissues by elevating temperatures to over 60 °C for a few seconds. On the other hand, HIFU mechanical ablation using histotripsy involves the creation of microscopic bubbles within the targeted tissue, which then collapse and generate mechanical shockwaves at much lower duty cycles to destroy the tissue. These microscopic bubbles can be created using ultrasound pulses in the range of microseconds to maintain stable or inertial cavitation bubbles in the tissue (cavitation histotripsy), or ultrasound pulses in the range of milliseconds to cause rapid and localized formation of vapor bubbles and heating of the tissue (boiling histotripsy, 100 °C; [8, 9]. Millisecond scale heating of 100 °C during boiling histotripsy prevents heat diffusion and damage of collateral tissues and only negligibly affects total temperature accumulation in the target regions, thereby avoiding denaturation of tumor antigens [10].

Both thermal and mechanical ablative techniques are known for their high precision in treating soft tissues, and immune activation. For example, mechanical lysis of murine breast tumors and neuroblastoma using boiling histotripsy (HT) was shown to enhance immunogenic cell death and formation of non-denaturing antigen depots resulting in superior therapeutic outcomes [10, 11]. Similarly, HIFU thermal ablation of murine tumors (e.g. neuroblastoma, hepatoma, etc.) and human prostate, osteosarcoma, hepatocellular carcinoma, and renal cell carcinoma tumors have shown to improve lymphocyte cytotoxicity [3, 12], and expression of heat shock proteins [13]. Although the utility of HIFU as a suitable non-invasive modality for solid tumor therapy is increasingly being recognized, the comparative assessment of HIFU exposures (mechanical and thermal) in enabling their translation to veterinary or human patients is largely unexplored. To address, the objective of this were two-fold (Fig. 1). First, we evaluated the ability of thermal and mechanical ablation using HIFU to locally target and achieve tumor control and anti-tumor immune effects in canine cancer patients, and then we assessed its ability to enhance tumor chemotherapy with mild hyperthermia (40–45 °C). Unlike ablative approaches that damages the tumor vasculature, and subsequently the flow of chemotherapeutics to the tumor, mild hyperthermia is the standard approach in preclinical studies to improve drug delivery [14–16].

Fig. 1.

Clinical trial study design. A Canine patients were screened to confirm the presence of a cancerous mass by physical exam, radiological and histological means. B They then were divided into the HIFU alone or HIFU + Dox cohort. Following HIFU exposures, tissue samples were collected immediately for drug delivery analysis. Immunological correlates were determined in blood samples 1–2 week post-treatment. Additionally, the patients were also monitored longitudinally for local tumor control

We utilized dogs with spontaneous solid tumors for our clinical trials due to their histologic similarity to human tumors [17]. Early trends from our laboratory and related reviews indicate that translating HIFU against tumors that are easily accessible (e.g. sarcoma, head and neck tumors) will provide novel mechanistic insights into its translational potential [18, 19]. Canine patients can also be longitudinally monitored for hematological, biochemical, and immunological parameters, making clinical trials in client-owned dogs immensely valuable. According to the American Veterinary Medical Association, one in four dogs will develop neoplasia spontaneously during their lifetime, which makes HIFU not only an innovative alternative treatment option for veterinarians and pet owners but also a means of accelerating human translation. Additionally, the ability of HIFU to be combined with conventional chemotherapy treatments can be suitable for large clinical trials. Therefore, the goals of our pilot veterinary trial are aligned with the objectives of current HIFU-based clinical trials, providing valuable insights to support the advancement of this treatment modality for patient use.

Materials and Methods

Patient Enrollment, Study Design, Inclusion/Exclusion Criteria, and Endpoints

All animal related procedures were approved by the Oklahoma State University Animal Care and Use Committee. Thoracic radiographs were also taken to confirm the absence of metastasis at the time of enrollment. Surgically resected patients with recurrence were not excluded from the HIFU trial. Prior to treatment, owner consent to the terms of the study, including a release from institutional/personal (researcher) liability, were obtained (Fig. 1). Canine cancer patients (n = 5patients/group) were assigned randomly into two treatment groups regardless of their tumor types as follows: (1) Thermal ablation and (2) histotripsy. The primary endpoint was local tumor control. Secondary endpoints were immune cell characterization 1 week post-HIFU exposure in blood by flow cytometry and profiling of serum cytokines. For HIFU + Dox combinatorial trials, three biopsy-positive sarcoma patients were enrolled and treated. The primary endpoint was Dox delivery analysis in the heated and unheated regions of the tumor. Secondary endpoints were therapeutic response characterizations, and assessment of immunological correlates by flow cytometry and cytokine analysis in blood and biopsy samples 2-week post-treatment. Unlike HIFU alone trials, in the combinatorial trial, Dox injections were performed by IV route, thus we chose to perform immune evaluations after 2 weeks to allow sufficient time for the regeneration of immune cells following the cytotoxic therapy.

HIFU Setup and Treatment

HIFU treatment was performed in anesthetized patients using an ultrasound-guided Alpinion system (Bothell, WA, USA) (Fig. 2A). For acoustic coupling, the dog’s fur was shaved at the treatment site to ensure complete contact with the transducer membrane. Degassed gel was used to provide acoustic coupling. The Alpinion planning software was used to select the region of interest and target boundaries in the X, Y, and Z directions for automatic rastering of the transducer. HIFU parameters (Table 1) used were previously optimized in vivo mouse studies and were similar to those reported in publications from our and other labs [18, 20]. Each patient experienced single HIFU exposure for thermal ablation and 1–2 for histotripsy (Table 2). ~ 25–50% of the total tumor volume was treated thermally or with histotripsy to allow immune cell infiltration in tumors.

Fig. 2.

An ultrasound-guided high-intensity focused ultrasound system (HIFU) was used for the dog treatments. A The figure inset shows the treatment transducer enclosing the probe, which was filled with distilled water and covered with a membrane. The membrane could be inflated to adjust for the depth at which the target lesion needs to be treated. A planning software was used to select a region of interest within the tumor for HIFU treatment. B An example of HIFU transducer coupling with the tumor prior to treatment (red ellipse)

Table 1.

HIFU treatment parameters used for tumor exposures in client-owned canine patients with spontaneous cancers

Parameter	Duty cycle (%)	Power (W)	Pulse repetition frequency	Treatment time/focal spot (s)	% of total tumor volume treated
Ablation	50	70–90	20	30–35	~ 50–60%
Histotripsy	1	450–600	5	20–25	~ 30–50%
Hyperthermia	50	8–12	20	300–420	~ 30–40%

Table 2.

Ablation patient characteristics and response evaluation using RECIST guidelines demonstrated partial to complete remission in majority of treated patients

Patient #	Breed	Age	Gender	Tumor location	Diagnosis	HIFU#	Response post HIFU treatments			Side effects
							Day 7	Day 14	Day 60
1	Lab retriever	6 years	FS	Tail base	MCT, low	1	CR	CR	CR	Skin inflammation and open wound were noted after tumor regression. Prescribed topical antibiotics and complete healing of wound was noted within a week
2	Great Pyrenees	9 years	MI	Right inguinal	Mammary massa	1	PR	CR	CR
3	Yorkshire mix	13 years 5 months	FS	Below the left sacral region	Apocrine gland carcinoma	1	CR	CR	CR	Owner reported diarrhea. Open wound was surgically closed and healed well
4	Mixed	6 years	MC	Right elbow region	Fibroma	1	CR	CR	CR	Thermal burn lesions were noted. Wound showed complete healing within 2 weeks post-surgical closure
5	French bulldog	8 years	MC	Left perineal region	MCT, low	1	PR	PD	–	Treatment area showed inflammation. Inguinal lymph node swelling reduced post treatment

Rx treatment, OR overall response, DFS disease free survival, CR complete response, PR partial response, PD progressive disease, SD stable disease, FS female spayed, FI female intact, MC male castrated, MI male intact, MCT mast cell tumor

^aReceived corticosteroid therapy for chronic arthritis prior to enrollment

Dox Administration and its Estimation in Tumors Using HPLC

Dox was injected at a dose of 0.7 mg Dox/kg body weight dispersed in D5W (5% dextrose in water) intravenously over 30 min after initiation of HIFU in the patients as published [21, 22]. Biopsies were taken immediately post-treatment from the heated and unheated parts of the tumor for Dox analysis. High-performance liquid chromatography (HPLC)/spectrophotometric analysis was performed using our previously published methods [16, 23]. Data were acquired using Shimadzu LC solution software (Kyoto, Japan). Tissue analyte concentrations were calculated using peak-area ratios of the sample analyte to the internal standard from the calibration curve [22].

Therapeutic Efficacy Assessments

Treatment efficacy was evaluated by tumor caliper measurements and recording of new lesions according to the Veterinary Cooperative Oncology Group Response Evaluation Criteria In Solid Tumors (RECIST v1.1) guidelines [24]. Complete response was defined as disappearance of all target lesions, partial response as a > 30% decrease in the longest tumor diameter, and stable disease as a < 30% decrease in tumors or < 20% increase in the longest diameters of target lesions after HIFU treatments. We defined progressive disease as either the appearance of one or more new lesions or at least a 20% increase in the longest diameter of target lesions.

Evaluation of Systemic Cytokine Levels Using Luminex xMAP Technology, and their Correlation with Therapeutic Efficacies

The multiplexing analysis of canine cytokines, chemokines, and growth factors was performed using the Luminex^™ 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta) with their canine Cytokine 13-Plex Discovery Assay^® (MilliporeSigma, Burlington, Massachusetts, USA). The 13-plex consisted of GM-CSF, IFNγ, IL-2, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-15, IL-18, IP-10/CXCL10, KC-like, MCP-1/CCL2, and TNFα with an assay sensitivity from 3.2 to 21.0 pg/mL. Amongst these, GM-CSF, IL-6, IL-7, IL-8/CXCL8, IL-15, IL-18, KC-like, and MCP-1/CCL2 was successfully detected in the pre-treated serum samples of all patients, and thus these were compared with post-treated collected samples for analysis purposes. Data fold changes were calculated using the formula: (Post-treatment levels/Pre-treatment levels)-1, and were plotted for the responder (complete, partial, or stable response) and non-responder (progressive disease) patients.

Immunophenotyping of Immune Cells with Flow Cytometry

For flow cytometry, blood samples were collected in BD Vacutainer EDTA tubes, and biopsy samples were placed in RPMI supplemented with 2% fetal bovine serum. Single-cell suspensions were obtained through mechanical disruption of the tumor biopsy tissues followed by enzymatic digestion with 200 U/ml of collagenase IV (Life Technologies, Grand Island, NY, USA) at 37°C for 1 h. The lysates were filtered through a 70 μm cell strainer (Corning Inc., Corning, NY, USA). Blood samples were incubated with 1× red blood cell lysis buffer (multi-species, Invitrogen, Waltham, MA, USA) for 10–15 min before antibody staining. The following fluorochrome-conjugated anti-dog antibodies were used to stain immune cells for 30 min in the dark on ice: anti-CD3⁺, anti-CD4⁺, anti-CD-8⁺ (dog T lymphocyte cocktail, cat. 558699, BD Pharmingen, San Diego, CA, USA), and APC-labeled anti-CD45⁺ (YKIX716.13, cat. MCA1042, Bio-Rad, Hercules, CA, USA). To detect IFN-γ and Foxp3⁺ T regulatory (Treg) cells, the cells were washed after surface marker staining, fixed, permeabilized with a transcription factor buffer set (BD Biosciences, San Jose, C, USA), and incubated with Alexa Fluor 700 labeled anti-IFN-γ (CC302, Novus Biologicals, Littleton, CO, USA) and e-fluor 450 labeled anti-Foxp3 (FJK-16 s, cat. 5016374, Fisher Scientific, Waltham, MA, USA) for 50 min in the dark on ice. Stained cells were analyzed using a FACS Aria II device (BD Biosciences) within 24–48 h. Compensation was performed with single-stained UltraComp eBeads (Invitrogen).

Statistical Analysis

Datasets were analyzed using FlowJo software v.10.2 (Treestar Inc., Ashland, OR, USA). Treatment groups were compared using two-tailed pairwise t-test (p values less than 0.05 were considered significant and represented as *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001). Since the thermal ablations and histotripsy patients were randomly distributed to the two treatment groups regardless of their tumor types, we utilized a harmonic mean-based evaluation approach to understand the association of therapeutic responses on day 7 with the flow cytometry outcomes. This approach has been widely adopted for performance evaluation in data analytics [25]. To do so, the populations of immunosuppressive Tregs (CD3 + CD4 + FoxP3 +) and immunoactivating CD3 + CD4 + or CD8 + IFN-γ T cells were combined into a single metric by taking their harmonic mean employing the equation given below [26]. Our analysis assumed that patients that responded to treatments had a reduced population of Tregs, and relatively higher populations of IFNγ + T-cells. The range of immune activations was computed by normalizing the data from 0 to 1. A higher value of harmonic mean indicated higher population of IFNγ + and lower FoxP3 +.

$$\begin{gathered} \text{Asssociation}\text{between}\text{therapeutic}\text{response}\text{and}\text{CD}4 \\ +\text{Tcell}\text{activation}=2\frac{(100-\text{CD}3+\text{CD}4+\text{FoxP}3+)\%\times \text{CD}3+\text{CD}4+\text{IFNg}+\%}{\left(\right(100-\text{CD}3+\text{CD}4+\text{FoxP}3+)\%+\text{CD}3+\text{CD}4+\text{IFNg}+)\%)} \end{gathered}$$

$$\begin{gathered} \text{Asssociation}\text{between}\text{therapeutic}\text{response}\text{and}\text{CD}8 \\ +\text{Tcell}\text{activation}=2\frac{(100-\text{CD}3+\text{CD}4+\text{FoxP}3+)\%\times \text{CD}3+\text{CD}8+\text{IFNg}+\%}{\left(\right(100-\text{CD}3+\text{CD}4+\text{FoxP}3+)\%+\text{CD}3+\text{CD}8+\text{IFNg}+)\%)} \end{gathered}$$

Results

HIFU Thermal Ablation Induced Tumor Remission in the Majority of Treated Patients

For ablation treatment, dogs with mast cell tumors (MCTs), mammary, apocrine gland carcinoma, and fibroma were included. The histotripsy trial included patients with mammary, mammary papillary adenocarcinoma, soft tissue sarcoma, lipoma, and low-grade MCT. Tables 2 and 3 summarize the patient characteristics, response rates, and adverse findings following thermal ablation and histotripsy per RECIST guidelines. Tumor volume changes were also compared using length*width²/2 over 7–14 days post-treatment. Overall, most patients treated with thermal ablation (n = 4/5) exhibited a complete response within 2 weeks after the first treatment (Fig. 3A and F). In subsequent follow-ups up to 60 days, these patients remained tumor free (Table 2). One of the five patients with an MCT showed an initial response at 1 week follow-up and remained in a stable disease state in the next follow-up. The owner decided to discontinue the treatment, and additional follow-up could not be performed. Histologically, H&E analysis of biopsy masses post-thermal ablation performed by a veterinary pathologist primarily showed necrosis characterized by loss of cellular details and inflammation in the HIFU exposed regions (Fig. 3C). Treatment-related adverse effects consisted mostly of moist dermatitis, minor burns, open wounds, and skin inflammation in the treated regions. In patients with fibroma and apocrine gland carcinoma with tumors close to attenuating structures (e.g., bones), local tissue damage due to thermal exposures required surgical suturing to close the open wounds post tumor-remission.

Table 3.

Histotripsy patient characteristics and response evaluation using RECIST guidelines indicated partial response to stable disease in the patients

Patient #	Breed	Age	Gender	Tumor location	Diagnosis	HIFU#	Response post HIFU treatments			Side effects
							Day 7	Day 14	60 days
1	Dachshund	–	FS	Left mammary mass	Mammary mass	1	PR	PR	–	None reported
2	Mixed	8 years	FS	Left mammary chain	Papillary adenocarcinoma	2	PD	PD	PR	None reported
3	Mixed	12 years	MC	Oral-right and left upper	Low grade soft tissue sarcoma	1	SD	SD	PD	None reported
4	German Shepherd dog mix	7 years 7 months	FS	Below the sacral regions	Lipoma	2	PR	PD	PD	None reported
5	Pit bull	8 years	MI	Left antebrachium	Recurrent MCT, lowa	2	PD	PD	PD	None reported

Rx treatment, OR overall response, CR complete response, PR partial response, PD progressive disease, SD stable disease, FS female spayed, MC male castrated, MI male intact, MCT mast cell tumor

^aMass recurred after surgical resection

Fig. 3.

Treatment responses to HIFU-mediated thermal ablation vs. HIFU non-thermal histotripsy exposures. A, B Representative images showing changes in tumor volumes with thermal ablation and histotripsy protocols, respectively. C Histopathology images of biopsy tissues show a marked increase in areas of necrosis (black arrows) characterized by loss of cellular detail, inflammation, and cell debris. D, E The hyperechoic regions with histotripsy matched the histological necrosis profile. F Clinical response with ablation and histotripsy was determined using both RECIST criteria (Tables 2, 3), and comparison of tumor volumes using the formula (length × width2)/2, where length was the largest dimension and width was the smallest dimension perpendicular to the length. Most patients treated with ablation showed complete remission within 1–2 week post-treatment (green). One patient showed partial remission and exhibited progressive disease (red). G From the histotripsy treated cohort, two dogs showed partial regression, one showed stable disease, and the remaining two patients showed progressive disease

Histotripsy was Well Tolerated and Achieved Partial Remission to Stable Disease

HIFU histotripsy induced tumor cell lysis, as demonstrated by real-time bubble cloud formation in the exposed regions imaged using US (Fig. 3D). Two out of five patients had tumors of mammary origin, which showed partial remission after treatment. One patient showed stable disease, and the other two treated patients demonstrated partial response followed by progressive disease (Table 3, Fig. 3B and G). In contrast to thermal ablation, histotripsy treatments were generally well tolerated by all patients. No damage to tumor-adjoining healthy tissues or systemic reactions was reported by the owners for any of the treated patients. Histologically, H&E analysis of biopsy masses performed by a veterinary pathologist post-histotripsy showed areas of necrosis, inflammatory influx, and cellular debris in the HIFU exposed regions (Fig. 3E).

Changes in Anti-tumor Cytokine Serum Levels Correlated with HIFU-Treatment Efficacy

In ablation group, patients who responded to the treatment showed a decrease in levels of GM-CSF, IL-7, IL-18, and MCP-1 and an increase in IL-8 on day 7 relative to pre-treatment levels (Fig. 4). Whereas in non-responders, these cytokines either didn’t change or increased compared to baseline levels. The decrease in IL-15 levels from the pre-treatment baseline was higher in non-responder compared to responders. Responders of histotripsy treatment showed a significant decrease in IL-6 and IL-15 levels from pre-treatment baselines compared to non-responders. Similar pattern was observed in levels of other pro-tumor cytokines, GM-CSF, IL-7, and IL-18 in responders compared to non-responders. MCP-1 levels increased in non-responders from pre-treatment baseline but remained unchanged in responders. An increase in IL-8 levels was observed post-histotripsy treatment in all patients.

Fig. 4.

Circulating levels of cytokines in serum from canine cancer patients (n = 5/group) treated with thermal ablation or histotripsy. Patient sera collected before and 7 days post HIFU exposure demonstrated trends in cytokine profiles that matched an augmented anti-tumor immune profile in patients who responded to the treatment. Non-responders demonstrated an increase in MCP-1, GM-CSF, and IL-7 levels in both histotripsy and ablation cohorts. Fold changes in serum cytokine and chemokine levels of each patient were calculated on the baseline of pre-treatment levels. (Two-tailed unpaired T-test, *p < 0.05.)

Histotripsy and Ablation Systemically Enhanced the Population of Activated T cells

Histotripsy didn’t induce changes in CD4 + and CD8 + T cell levels in the blood relative to the pre-treatment levels on Day 7 in almost all patients (Fig. 5A, D). In contrast, thermal ablation enhanced circulating CD4 + T cells on Day 7 (Fig. 5A) and slightly decreased CD8 + T cells (Fig. 5D). A trend of enhanced IFN-γ expression was seen for CD4 + and CD8 + phenotypes in both ablation- and histotripsy-treated patients (Fig. 5B, E). However, a clear statistical significance with flow cytometry analysis was not observed with pairwise comparison in patients. Next, we correlated the trends of immune profiles using harmonic mean index wherein higher populations of IFN-γ T-cells and reduced numbers of Tregs with therapeutic efficacy were observed. 4 out of 5 thermally ablated patients showed an increase in CD4 + (Fig. 5C) and CD8 + (Fig. 5F) activity, and CD8 + associated treatment response. In contrast, three out of the five patients showed an enhanced harmonic mean index for histotripsy, with one of the patients for CD4 + (Fig. 5C) and 2 out of 5 patients for CD8 + showing prominent association with the therapeutic responses (Fig. 5F).

Fig. 5.

Immunological analyses of blood samples showed enhanced IFN-γ-producing T cells post treatment with HIFU alone. Representative bar plots showing the differences in immune responses in ablation- vs. histotripsy-treated patients 7 days post-treatment, demonstrated by A and D circulating T cells that showed no changes in CD4 + and CD8 + T cells in histotripsy group, but an increase in CD4 + T cells and subsequent decrease in CD8 + T cells in ablation group; B and E IFN-γ production by T cell subsets showed higher expressions in CD4 + and CD8 + cells for the ablation and histotripsy cohort post-treatment; C and F Harmonic mean index showed correlation of high IFN-γ expressing T cells and reduced Tregs with favorable clinical outcomes in the treated patients. The upper dashed line represents the initial tumor volume. The initial T cell number for each dog is denoted by the red (histotripsy) and blue (ablation) circles on the dashed line. An increase or decrease in the relative tumor volume post-treatment for each patient is shown by the lines ending in an arrow. The increase in the functional T cells for the patients is shown by arrows moving toward the far-right x-axis, and the reduction in the population is shown by the movement of lines toward the starting point (0). The harmonic mean-based index typically increased with the decrease in tumor volumes of patients. (Two-tailed paired T-test, *p < 0.05.)

Adding HIFU to Dox Therapy Increased Targeted Drug Delivery and Efficacy

In dogs treated with HIFU + Dox, the drug concentrations in the heated regions (0.325 ± 0.01 μg Dox/g) of tumors were significantly higher than the unheated regions (0.26 ± 0.02 μg Dox/g; Fig. 6A). All three patients demonstrated tumor regression at 1-week post-treatment compared to the pre-treatment volumes (Fig. 6C). One of three patients showed regrowth of tumor in the second-week post-treatment. Also, progressive disease was noted in all cases on day 60 (Table 4).

Fig. 6.

HIFU enhanced Dox delivery to the heated regions and tumor regression. A Treatment regions in the tumor were divided into heated and unheated parts and biopsy samples were collected from the farther ends of the respective regions for estimation of Dox delivery using HPLC. B Comparison of μg Dox/g of tumor for heated and unheated regions of tumors following a 0.7 mg/kg body weight injection of Dox alone. Dox delivery to the heated regions was significantly higher than delivery to the unheated paired controls. Data are shown as mean Dox concentration ± standard error of the mean (n = 3; pairwise comparison; *p < 0.05, Two-tailed t-test). C Tumor volumes represented as fold change post-treatment with the HIFU + Dox combination showed regression in all three patients at 1–2 week post-treatment

Table 4.

Hyperthermia + Dox treated patient response evaluation using RECIST guidelines showed tumor remissions post treatment in all patients

Patient #	Breed	Age	Gender	Tumor location	Diagnosis	Route	HIFU#	Response post HIFU treatments			Side effects
								Day 7	Day 14	Day 60
1	Dachshund	11 years	MC	Right ventral chest	STS, grade I	I/V	1	PR	PR	PD	None
2	Boxer	8 years	MC	Right antebrachium	Recurrent STS, grade Ia	I/V	1	PR	PR	PD
3	Mini schnauzer	9 years 6 months	FS	Right shoulder	Recurrent STS, grade Ia	I/V	1	PR	PD	PD

Rx treatment, OR overall response, CR complete response, PR partial response, PD progressive disease, SD stable disease, FS female spayed, MC male castrated, STS soft tissue sarcoma, I/V intravenous

^aMass recurred after surgical resection

Adding HIFU to Dox Chemotherapy Augmented the Anti-tumor Immune Effects

HIFU + Dox treatment significantly increased CD3 + T cell levels in the blood (Fig. 7A) and increased infiltration (~ three fold) in tumors post-treatment relative to the pre-treatment levels (Fig. 7B). An increase in IFN-γ expressing CD4 + and CD8 + T cells and concurrent high ratios of circulating CD8:Tregs relative to the pre-treatment paired control levels at Day 7 post-treatment was noted (Fig. 7A, B). The serum cytokine levels corresponded with the populations of functional immune cells, with the post-treatment levels of GM-CSF, IL-6, IL-15, and IL-18 cytokines showing reductions in the serum levels post-treatment (Fig. 8), while MCP-1 and IL-8 demonstrated a spike compared to baseline.

Fig. 7.

Immunological analyses of blood and biopsy samples showed modulation of T lymphocyte response in Dox + HIFU treated patients. A, B A significantly higher population of CD3 + T cells with high IFN-γ expression with a relatively higher CD8:Treg ratio was observed on Day 7 in blood and day 14 in biopsy samples. (*p < 0.05, Two-tailed Paired t-test)

Fig. 8.

Circulating levels of serum cytokines pre- and post-treatment in canine sarcoma patients (n = 3) treated with Dox + HIFU (~ 40–45 °C). Trends in cytokine profiles showed decreased production of cytokines aiding tumor progression (e.g. IL-18, IL-15, IL-7, IL-6, etc.). A concurrent increase of pro-tumoral cytokine (e.g. IL-8, MCP-1) post-treatment was also noted. (****p < 0.0001, Two-tailed Paired t-test)

Discussion

Pre-clinical data from our laboratory and others have extensively established HIFU’s feasibility in murine and rabbit models [16, 22, 27, 28], but little is known about how these findings relate to actual clinical situations. Herein, we conducted pilot canine trials in patients with spontaneous tumors to evaluate the effectiveness of HIFU-based thermal ablation and compared those with mechanical histotripsy. We chose to conduct our trials in canine patients because tumors in dogs spontaneously occur, as they do in humans [29, 30]. For our HIFU monotherapy trials, we enrolled patients independent of tumor type. We made this choice based on the premise that cytotoxic exposures would have killed cancer cells regardless of their multiplication and drug resistance status. In contrast, we used dogs with soft tissue sarcoma for the combinatorial trials of Dox with HIFU because these tumors are known to be sensitive to anthracycline agents [31–33].

Each dog-patient underwent HIFU treatments covering ~ 25–50% of the total tumor volume. We hypothesized that partial ablations would have prevented damage to peripheral vasculature system of tumors, thereby aiding the infiltration of cytotoxic immune cells in the treated masses. Also, relative to whole gland ablation, partial ablation in prostate cancer patients has been shown to be associated with improved clinical outcomes, especially in terms of the adverse effect profiles (e.g. urinary incontinence) [34]. Overall, the tumor remissions from thermal ablation were relatively more effective compared with those of histotripsy treatment (Table 2; Fig. 3). Four patients showed complete remission up to 60 days, demonstrating effective outcomes of thermal ablations. In the two patients with MCTs, one responded to the ablation treatment, whereas the other showed a partial response followed by a progressive disease. Garrett previously reported that dogs with lymph node metastasis with MCT demonstrated shorter median survival times than dogs without nodal metastasis [35]. The non-responder patient in our study presented with an inguinal lymph node metastasis at the time of diagnosis, and we believe that it likely influenced the therapeutic outcomes due to a more aggressive variant of MCT.

Thermal ablation of tumors that are situated in close proximity to bony structures can cause tissue wounds due to high acoustic absorption of sound waves. For instance, Kopelman et al. performed magnetic resonance (MR)-guided HIFU of hepatocellular adenoma in a canine patient and reported focal thermal injury around the ribs post-treatment [36]. We also noted minor burns and ulcerations in the treated regions, but we managed them using hyperbaric oxygen therapy and antibiotics and with surgical closure of open wounds post-tumor remission [18]. As an alternative, we investigated the utility of non-thermal tumor histotripsy over thermal ablation. Prior studies in canines that underwent histotripsy of healthy prostate tissues and sarcoma yielded acellular disruption of targeted tissues [37–40]. In our trials, histotripsy similarly induced remission, but the reduction in tumor volumes was relatively lower compared to thermal ablation (Table 3 and Fig. 3). Based on this outcome, we believe that histotripsy prior to surgery can potentially reduce the risk and morbidity associated with resection of tumors. It may be noted that high-grade mast cell tumors and soft tissue sarcomas can recur one year after surgical resection [41–43]. Thus, the 60-day follow-up in our trials may be limited, and future HIFU trials can aim to follow patients for > 1 year to provide a more realistic evaluation of treatment success.

Immunologically, both histotripsy and thermal ablation increased IFN-γ expressing CD3 + CD4 + and CD3 + CD8 + T cells in the blood compared with the pre-treatment levels (Fig. 5). In general, the immune activation trends for CD8 + T cells were relatively more prominent with histotripsy exposure compared to thermal ablation. Histotripsy generates subcellular fragments through mechanical fractionation, thereby releasing damage-associated molecular patterns that enhance tumor inflammation and anti-tumor immune effects [44, 45]. The induced danger signal production can depend on the tumor type, with certain types (colon adenocarcinoma, renal cell carcinoma, and breast adenocarcinoma) showing better activation than others (e.g. neuroblastoma) [46]. Our assessment of the systemic cytokine/chemokine responses in serum suggested that GM-CSF, IL-7, IL-15, and IL-18, were downregulated by both histotripsy and ablation in patients who responded to the HIFU treatment (Fig. 4). GM-CSF is produced by activated immune cells, and several studies have shown that it can promote tumor progression [47]. Similarly, an increase in circulatory levels of IL-7, IL-15, and IL-8 is known to be associated with the progression of prostate, melanoma, and non-small lung cancers [48, 49]. Our data found a consistent decrease in serum IL-18 with both types of HIFU treatments in responders and relatively higher serum levels of MCP-1 in non-responders. MCP-1 is produced by immune cells (e.g., monocytes), endothelial cells, and several malignant tumors. It increases angiogenesis and promotes suppressive TME [50]. Future studies focused on a single tumor type incorporating anti-MCP-1 and/or IL-8 interventions can shed more light on whether improved therapeutic outcomes in non-responders can be attained with such an approach.

A second major objective of our pilot trial was to evaluate the ability of HIFU to improve Dox delivery to tumors (Table 4). Addition of hyperthermia to intravenous Dox chemotherapy significantly enhanced drug delivery in the heated regions of the tumor (Fig. 6B), and associated with tumor regression and improved anti-tumor immune effects (e.g. high anti-tumoral CD8 + T cell subsets in the tumor biopsies and blood; Fig. 7A, B). Dox is known to induce immune mediated tumor remission via the expression of calreticulin (CRT), high mobility group box protein 1 (HMGB1), heat shock proteins, and ATP leading to immunogenic cell death (ICD) [51–53]. Our data showed significant enhancement in the infiltration of T cells in the tumor biopsy masses post-HIFU treatment (Fig. 7B), however, with only a single treatment, the tumors started to grow back in all three patients during subsequent follow-ups in week 2 and later. This suggests that multiple cycles of Dox plus HIFU treatment are needed to achieve significant control of sarcoma growth. Also, for our trials, ~ 40% of the tumor mass of each patient received local hyperthermia treatment. We propose that heating the entire tumor volume would achieve higher Dox levels and therapeutic responses compared to partial tumor heating. Lastly, we noted that the serum IL-8 and MCP-1 levels were elevated with Dox + HIFU post-treatment, and this may have also contributed to tumor regrowth (Fig. 8). Interestingly, we also observed a slight increase in serum IL-8 levels in responders of thermal ablation and histotripsy treated patients. IL-8 cytokine is often associated with immunotherapy responses in a variety of solid tumors in human patients [54]. Several preclinical studies have also reported an increased IL-8 levels in mice undergoing tumor remission with histotripsy and thermal ablation [4, 10, 55]. Thus the observed increase of IL-8 in responder patients might be due to the thermal and mechanical damage inflicted on tumor tissue, leading to a compensatory fibrotic and healing response as reported previously [56]. Unlike ablations, mild hyperthermia doesn’t cause any tissue damage, thus it likely didn’t contribute towards the serum cytokine elevations. Future trials comparing the short and long-term cytokine responses following multiple cycles of Dox + HIFU can provide more insights into the clinical advantages and feasibility.

In summary, depending on HIFU parameters, tumor growth control with moderate to no adverse effects can be attained in canine cancer patients. Considering the improved anti-tumor immune effects conferred by thermal ablation/histotripsy, they can be combined in future trials, as the combination may achieve superior therapeutic outcomes. HIFU also allows greater Dox delivery to tumors, which correlates with improved local and systemic anti-tumor immunity and tumor regression. Additional studies in a larger cohort of patients of similar tumor types will provide more insights into the ability of this approach to improve survival and anti-tumor immunity.